Centriole biogenesis, and the duplication cycle, amazing evidence of design

Centriole biogenesis, and the duplication cycle, amazing evidence of design

https://reasonandscience.catsboard.com/t2090-centriole-biogenesis-and-the-duplication-cycle-amazing-evidence-of-design

The  duplication of eukaryotic cells is a all fine-tuned biochemical processes that depends on the precise structural arrangement of the cellular components. Mitotic cell division is the most fundamental task of all living cells. Cells have intricate and tightly regulated machinery to ensure that mitosis occurs with appropriate frequency and high fidelity.

The only way to make a new cell is to duplicate a cell that already exists. A cell reproduces by performing an orderly sequence of events in which it duplicates its contents and then divides in two.  This cycle of duplication and division, known as the cell cycle, is the essential mechanism by which all living things reproduce. Dividing cells must coordinate their growth. A complex network of regulatory proteins  trigger the different events of the cycle. 

During the cell cylce, eighteen different regulators are required, which order and coordinate the process. Each of these regulators are absolutely essential. If one is missing, the cell cycle is not completed and, the cell cannot duplicate.  Any of these regulators have only use if fully integrated in the process. They have no use or function by themself. This makes  replication a irreducible , interdependent process.

Centrosomes play a key role in organizing the microtubule network of the cell, most notably the mitotic spindle during cell division .

The choreography of microtubules, centrosomes and chromosomes during mitosis and meiosis is beautifully designed, and uses finely regulated and synchronized movements.

The centrosome is a structure, consisting of a pair of cylindrical microtubule-based organelles called centrioles , embedded in an amorphous network of proteins known collectively as Pericentriolar Material (PCM). Microtubules (MTs) originate from the PCM.  The PCM comprises a porous structural scaffold onto which γ-tubulin and other soluble components from the cytoplasm are loaded. Centrosome growth is an aggregation process of a condensed phase of PCM components, which segregate from the cytosol. The aggregation process leads to a centrosome phase that coexists with the cytosol and does rearrange internally. This implies that the centrosome phase is viscoelastic, such that on long timescales it behaves as a liquid-like material.

Cep192 is a pericentriolar protein that accumulates at centrosomes during mitosis and is required for PCM recruitment, centriole duplication, microtubule nucleation, and centrosome maturation.

Centrioles are among the most beautiful of biological structures. How their highly conserved nine-fold symmetry is generated is a question that has intrigued cell biologists for decades.
Centrioles are present in all eukaryotic species that form cilia and flagella, but are absent from higher plants and higher fungi which do not have cilia.
It seems likely that they have  the primary purpose of growing cilia and flagella, which are important sensory and motile organelles found in almost all cells of the human body.  These organelles have many important functions in cells, and their dysfunction has been linked to a plethora of human pathologies, ranging from cancer to microcephaly to obesity.  Great progress has been made recently in understanding how these proteins interact and how these interactions are regulated to ensure that a new centriole is only formed at the right place and at the right time.

Centriole biogenesis requires thirteen essential molecules. If any of these molecules is missing, centrioles cannot be made. 

Centriole assembly is also tightly regulated and abnormalities in this process can lead to developmental defects and cancer. Initiation of centriole duplication is under tight regulation to ensure the control of centriole number. Presumably in centriole initiation, there is some form of cooperativity or positive feedback that results in asymmetric accumulation of the relevant proteins in a symmetric background.

So we have not only the requirement of eighteen proteins required for cell cycle regulation, but also thirteen essential molecules for centriole biogenesis, which by itself is also tighthly regulated, requiring positive feedback.

It appears at the initial stage of the centriole assembly process as the first ninefold symmetrical structure. The cartwheel was first described more than 50 years ago, but it is only recently that its pivotal role in establishing the ninefold symmetry of the centriole was demonstrated.  This is a highly ordered structure that really stands out from the background. Constructed of rod-like microtubules, most centrioles have a nine-fold pattern, nine triplets or doublets evenly spaced at the rim, giving it a "cartwheel" appearance in cross-section. 

The comparison to a human made cartwheel is evident, and so that it is intelligently designed. Obviously, the question arises, how could all this emerge gradually ? 

Another amazing fact is that Electromagnetics play an important role in cell functioning and especially in cell duplication and division (mitosis).

Recent development in the field of quantum biology highlights that the intracellular electromagnetic field (EMF) of microtubules plays an important role in many fundamental cellular processes such as mitosis. It is an intriguing hypothesis that centrosome functions as molecular dynamo to generate electric flow over the microtubules, leading to the electric excitation of microtubule EMF that is required for spindle body microtubule self-assembly. With the help of motors proteins within the centrosome, centrosome transforms the energy from ATP into intracellular EMF in the living cell that shapes the functions of microtubules. There will be a general impact for the cell biology field to understand the mechanistic function of centrosome for the first time in correlation with its structural features.



The electromagnetic property of microtubule has been reported with both computation modelling and experimental evidences.

To transform the chemical energy in ATP into electric magnetic field within the living cell, cell needs to have a molecular dynamo to transform the mechanistic movement of protein complexes to directional movements of intracellular electrons, leading to the electric excitation of the spindle body microtubules as well as the M phase chromosomes, which is essential for mitosis

Taken together the longitudinal, or axial, vibration of the 13 filaments of an MT and then the 27 MTs making up the centriole barrel produce the electromagnetic field surrounding the centriole . Interestingly, this field is also found to be ferromagnetic. Also of interest, the fundamental vibration frequency of an MT filament is approximately 465 MHz, although this frequency is continually changing due to the ongoing length changing of the filaments. The electropolarity of the centrioles enables them to exert forces at a distance—that is, forces without physical contact.

"All this cannot [but] leave us astounded[; it] indicates the requirement of for[e]sight to produce all these ingenius[ingenious] mechanisms,and subsequently[consequently] a[n] intelligent designer.

Yuan Tian: Nine-fold symmetry of centriole: The joint efforts of its core proteins 07 January 2022
https://onlinelibrary.wiley.com/doi/abs/10.1002/bies.202100262



https://reasonandscience.catsboard.com/t2090-centriole-biogenesis-and-the-duplication-cycle-amazing-evidence-of-design

Centrioles, Centrosomes, and Cilia 7
The centrosome is the primary microtubule-organizing center (MTOC) in animal cells. It regulates cell motility, adhesion, and polarity during interphase of the cell cycle and
facilitates the organization of the spindle poles during mitosis. The centrosome comprises two centrioles that are surrounded by an electron-dense and protein-rich matrix
called the pericentriolar material (PCM). The canonical centriole has nine microtubule (MT) triplets and is ~0.5 µm long and 0.2 µm in diameter. The mother centriole has
subdistal and distal appendages, which dock cytoplasmic MTs and may anchor centrioles to the cell membrane to serve as basal bodies. Basal bodies seed the growth of
the axoneme, the structure that confers rigidity and motility to cilia and flagella. Cilia and flagella play critical roles in physiology, development, and disease. Most motile cilia
display axonemes that have 9 doublets and 1 central pair (A), whereas nonmotile, primary cilia display 9 doublets with no central pair (B). Abnormalities in centrosomes occur
in many types of cancer and can be associated with genomic instability. This is due to the fact that supernumerary and often irregular centrosomes can result in aberrant cell
division as well as abnormalities during asymmetric cell division.

Centriole Biogenesis
Components of the PCM, such as γ-tubulin, may play a role early in the process of centriole biogenesis. SAK/PLK4, a protein kinase of the Polo-like protein kinase family, is
essential for centriole biogenesis in flies and in human cells. This kinase is also known to be mutated in hepatocellular carcinomas; mice that have only one copy of the gene
encoding SAK/PLK4 are more prone to develop cancer. Overexpression of SAK/PLK4, or suppression of its degradation by the SCF/Slimb complex, leads to an increase
in the number of centrioles, with each mother centriole being able to nucleate more than one daughter centriole at a time. Most strikingly, this kinase can trigger centriole
formation de novo in Drosophila eggs or tissue culture cells depleted of centrioles.
The first described intermediate showing nine-fold symmetry in centriole assembly is the cartwheel. Bld10/CEP135 and SAS6 are two essential components of the cartwheel.
Mutations in those molecules most often result in failure to form centrioles or formation of centrioles with abnormal symmetry. Assembly and stabilization of centriole MTs
are dependent on SAS4 and γ-tubulin. Posttranslational modification of MTs may also play a role. Another component, CP110, may be essential for capping the centriolar
structure to regulate its length and function. Bld10, SAS6, and SAS4 all act downstream of SAK/PLK4 in canonical centriole biogenesis. SAS6 and SAS4 are also required
downstream of SAK/PLK4 in de novo centriole formation, suggesting a unique pathway for centriole biogenesis triggered by SAK/PLK4.

The Centrosome Cycle
The number of centrioles in a cell is controlled through a canonical duplication cycle that is coordinated with the chromosome duplication cycle. CDK1, CDK2, and Separase,
among others proteins, may play a role in coordinating the two cycles. During centriole duplication, one new centriole (daughter) forms orthogonally to each existing centriole
(mother) in a conservative fashion, once per cell cycle. Four consecutive steps in the centrosome cycle have been defined through electron microscopy: 

disengagement of the centrioles, 
nucleation of the daughter centrioles, 
elongation of the daughter centrioles, and 
separation of the centrosomes. 

Disengagement of centrioles is coordinated with chromatid segregation during mitotic exit and is required for duplication in the next cycle. Nucleation of daughter centrioles is coordinated with DNA synthesis, whereas centrosome separation occurs during G2 phase of the cell cycle. When the cell enters mitosis, it is equipped with two centrosomes that then participate in mitotic spindle assembly. SAS6 and SAK/PLK4 are tightly regulated during the cell cycle to prevent centriole amplification

http://www.cell.com/pb/assets/raw/journals/research/snapshots/PIIS0092867408016413.pdf

Centrosomes are conserved microtubule-based organelles that are essential for animal development


Centrosomes organize the microtubule cytoskeleton and are essential for the assembly of cilia in animal cells (Brito et al., 2012).



They are conserved microtubule-based organelles that are essential for animal development.  1 Centrosomes, the yeast equivalents of which are known as spindle pole bodies (SPBs), are microtubule-organizing centers of eukaryotic cells, which are made up of proteins but, like chromosomal DNA, are replicated in a tightly regulated manner that is coordinated closely with the cell division cycle. 2

Regulation and coordination requires complex specific precise informational input, which is based and requires intelligent action and programming.

Studies in C. elegans reveal an evolutionary conserved pathway in centriole formation (Pelletier et al., 2006; Delattre et al., 2006), and 


Several key components were identified in Drosophila melanogaster and human cells 


Spd-2/Cep192; 
Zyg-1/SAK/Plk4; 
Sas-6; 
Sas-5/Ana-2/STIL and 
Sas-4/CPAP) 


(Brito et al., 2012).

The mechanism of centrosome duplication is poorly understood, but insights are coming from studies of components such as centrin, a small calcium-binding protein that was first identified in the flagella of green algae . Centrins have turned out to be ubiquitous, widely conserved proteins, now known from a variety of studies to be involved in assembly, and in some cases maintenance, of centrosomes, SPBs and the basal bodies of flagellae. The involvement of centrin in multiple cellular processes, some calcium-dependent and some not, suggested that alternative binding partners would be discovered for the protein that define particular functions, and this has turned out to be the case. Several years ago, Kilmartin  uncovered a novel yeast protein called Sfi1 which binds centrin in the absence of calcium, is conserved in vertebrates and localizes to centrosomes in both yeast and vertebrates. Now, Kilmartin and colleagues have reported a structural analysis of the Sfi1–centrin complex and its asymmetric arrangement in the SPB (Figure 1), the results of which suggest a plausible model for the initiation, if not the licensing, of SPB duplication.

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. 3

Mutations in the budding yeast centrin, encoded by the CDC31 gene, or in the fission yeast homolog, SpCdc31, led to a block in the earliest step of SPB duplication and resulted in mitotic arrest with a monopolar spindle 1

They participate in mitotic spindle formation and thereby ensure faithful chromosome segregation during cell division. One centrosome consists of a pair of centrioles surrounded by the pericentriolar material (PCM). Like DNA replication, centrosome duplication is tightly regulated and restricted to only once per cell cycle. Thus, controlling centriole numbers ensures that cells have the proper number of centrosomes and cilia. Failures in centriole duplication result in abnormal centriole numbers, which have been linked to genomic instability and tumorigenesis. 5

Centriole biogenesis, and the duplication cycle 4

Centrioles are cylindrical cellular structures present in almost all eukaryotic lineages. 6,  with a signature morphological motif of nine specialized microtubules symmetrically arranged about a central core (figure 1a). A core set of five centriolar proteins has been identified and their sequential recruitment to procentrioles has been established. Centrioles usually exist in pairs, oriented perpendicularly to one another, and are surrounded by the pericentriolar material (PCM) to form the centrosome.

 The PCM has a layered structure made of fibres and matrix proteins, and is responsible for microtubule nucleation and anchoring. Mature centrioles are differentiated along their length for different functions. The distal end of the centriole is often specialized by the addition of appendages to organize microtubules and to nucleate a cilium [1,2], and interacts with the plasma membrane during ciliogenesis. The proximal end of the centriole is specialized to recruit a matrix of pericentriolar material proteins that support microtubule nucleation, and organization of microtubule arrays [3]. Centrioles are present in all eukaryotic species that form cilia and flagella, but are absent from higher plants and higher fungi which do not have cilia. Importantly, basal members of the plant and fungal groups do have centrioles and cilia, suggesting that these organelles are among the features that defined the earliest eukaryotic ancestor [4].



Centriole assembly pathway in vertebrates. 
(a) Centrioles are cylindrical structures composed of nine microtubule triplets symmetrically arranged about a central core. The components important to the discussion here are indicated in the legend. Depicted is a longitudinal section of a mother centriole, which has two types of appendages, distal and subdistal, and lacks the internal cartwheel structure. The base of the mother centriole is embedded in the pericentriolar material. The formation of a procentriole has been initiated by assembly of the stalk and cartwheel from the side of the mother centriole. 
(a: 1–4) Stages of procentriole formation, depicted as viewed by cross section of centriole at X in longitudinal section. The mother centriole is not shown in (a: 2–4) for clarity, but is present and engaged to the procentriole throughout the process shown. 
(a-1) PLK4 accumulates at a single focus, in conjunction with CEP152 and CEP192, which are distributed in rings around the circumference of the centriole. PLK4 stimulates the assembly of a stalk and ninefold symmetric cartwheel that will provide structure to the procentriole and keep it engaged to the mother centriole. 
(a-2) Nine A-tubules are nucleated by the gamma-tubulin ring complex (gamma-TURC), in association with the cartwheel. These grow unidirectionally from the proximal to the distal end of the centriole. The A-tubules remain capped by the gamma-TURC throughout the assembly process, eventually being lost at the end of mitosis. 
(a-3) The B- and C-tubules form by a gamma-TURC-independent mechanism and grow until they reach the length of the A-tubule. 
(a-4) The distal end of the centriole is formed by elongation of the A- and B-tubules, creating a structurally distinct distal domain. (b) Centriole disengagement in the transition from M-G1. (i) A centrosome in metaphase of mitosis, with engaged mother centriole and procentriole. (ii) A centrosome in G1, after mitosis, with disengaged mother and daughter centrioles. The cartwheel has disassembled from the daughter centriole. Note that the subdistal appendages disassemble during mitosis, but the constituent proteins remain associated with the centriole. They are depicted as undifferentiated spheres in the mitotic centriole in place of the subdistal appendages. (Online version in colour.)

The structural complexity of the centrioles is reflected in the large number of centriole proteins identified by a combination of genomic and proteomic approaches in the past decade [5–19]. Some of the centriole proteins are conserved in all organisms with centrioles [4,20,21]. By contrast, some centriole proteins are unique to a subset of organisms and tissues and these differences are probably owing to the diversity of contexts in which centrioles assemble and function. In dividing cells, centrioles duplicate once per cell cycle, adjacent to a pre-existing centriole. What specifies the structure of a centriole, its location, orientation and copy number has been a longstanding question since the initial observations of the events of centriole duplication. The ‘parts list’ for the centriole is probably nearly complete, and the more recent challenge has been to address how these parts fit together to assemble centrioles. In this review, we first discuss the underlying mechanisms for the morphological duplication of centrioles, and how the complex ultrastructure of centrioles with ninefold symmetry and a well-defined length is established. We next describe progress in our understanding of how cells ensure centriole copy number in successive cell division and also how these control mechanisms are modified in different contexts.

2. Morphological duplication of centrioles–centriole biogenesis

The structure of centrioles is remarkably conserved across the eukaryotic kingdom (figure 1a). All known centrioles are cylindrical in shape and are composed of a ninefold symmetric array of microtubules. However, among different organisms, these microtubule arrays can comprise singlet, doublet or triplet microtubules, and centriole size ranges from 100 to 250 nm in diameter and from 100 to 400 nm in length [4]. The morphological events of the centriole duplication cycle have been well defined through electron microscopy at the ultrastructural level [23–27] (figure 1). Centriole duplication begins at the G1–S transition, when a new daughter centriole, termed a procentriole, begins to grow orthogonally from the proximal end of each of the two existing centrioles, termed mother centrioles. Once formed, procentrioles elongate through S and G2 phases. At the end of mitosis, the mitotic spindle segregates the duplicated centriole pairs, so that each resulting daughter cell contains two centrioles. Here, we discuss what is known of how the morphological duplication of the defining features of centrioles is accomplished each cell cycle. The first step in the centriole duplication cycle is the formation of a procentriole adjacent to the mother centriole (figure 1a). This occurs at only one site per centriole, to ensure centriole number control.

To ensure this pressuposes intention and distant goal. 

We shall refer to this site as the origin of centriole duplication, by analogy to DNA replication. What defines the location of the origin of centriole duplication? Emerging evidence suggests that three centriole proteins, PLK4, SASS6 and STIL, have an instructive function in this process as they all localize at the site of procentriole assembly at the G1–S transition, when centriole assembly is initiated. SASS6 is a structural component of the cartwheel, the ninefold symmetric template structure internal to the proximal end of the centriole (see other chapter) [28–30], and STIL is associated with SASS6 [31–34].

Although SASS6 was previously reported to be the earliest marker at the site of procentriole assembly [35], two independent reports find that PLK4, a polo-like kinase required for centriole formation [36,37], localizes to a dot-like structure at this site prior to SASS6, suggesting that it recruits SASS6 [36,37] (figure 1a-1). In Caenorhabditis elegans, SAS-6 is recruited to the centriole by ZYG-1, a functional orthologue of PLK4, thus this might be an  conserved mechanism of defining the site of centriole assembly [38]. Strikingly, PLK4 at the centriole changes from a ring-like localization around the centriole barrel (early G1) to the dot-like localization (G1–S transition), and this change coincides with initiation of downstream events of centriole duplication, further supporting the role of PLK4 in marking the origin of centriole duplication [36]. These studies identify PLK4 as the earliest marker that localizes to the site of procentriole assembly, and given that PLK4 is a protein kinase, the most straightforward model would be that PLK4 phosphorylation of downstream proteins in centriole assembly initiates the process. Although some proteins have been shown to be substrates of PLK4 in vitro, including some centriole duplication proteins [39–42], there is as yet no direct link between PLK4 kinase activity and centriole initiation.

If PLK4 defines the origin of centriole duplication, what targets PLK4 to the centrioles at the right time and place? Recent studies suggest that two PLK4-binding proteins are important in this process. PLK4 (or ZYG-1) recruitment to the centrioles depends on Asterless in Drosophila [43] and on SPD-2 in C. elegans [44,45]. Interestingly, in mammalian cells, the orthologues of these two proteins, CEP152 and CEP192, respectively, interact with PLK4 and cooperate in the recruitment of PLK4 to the centrioles [36,46] (figure 1a-1).






This recruitment depends on electrostatic interactions between the positively charged polo-box domain of PLK4 and acidic regions of CEP192 and CEP152 [36,46]. Both CEP152 and CEP192 are distributed symmetrically around the circumference of the centriole barrel [37,47], thus, although these studies provide an appealing explanation for how PLK4 is recruited to centrioles, they do not address the fundamental question, which is how is a single site for initiation of a morphological event chosen from a radially symmetric surface? This is, in essence, a symmetry-breaking event, similar to that in choosing a single site for bud formation in budding yeast [48], or establishment of the axes of a C. elegans embryo [49,50]. In these cases, and presumably in centriole initiation, there is some form of cooperativity or positive feedback that results in asymmetric accumulation of the relevant proteins in a symmetric background.

Question: Is that not a essential process, that had to be set-up just right, and fully functional  from the start ? 

It is possible, then, that association of PLK4 with CEP192 and/or CEP152 has such cooperative properties, or that the kinase activity of PLK4 provides some positive feedback mechanism to that association. Importantly, such a model would depend critically on the concentration of PLK4 in the cell, and that the concentration be subsaturating with respect to its binding partners; as described below, the concentration of PLK4 is known to be critical to limiting duplication to one site.

Once the site of the origin of centriole duplication is defined on the mother centriole, the next step is formation of the cartwheel [22] (figure 1a-1). The ninefold symmetric structure of the cartwheel is derived from the intrinsic ninefold symmetry of SASS6 oligomers that form the cartwheel [28,30]. The mechanism of cartwheel formation is covered in another review in this theme issue. The cartwheel dictates the ninefold symmetry of centrioles and initiates sequential assembly of the nine triplet microtubules. Interestingly, in some cases (mammals included), the cartwheel is lost from mature centrioles [22,51], thus it is required to make, but not to maintain, the structure of the centriole. Cryoelectron tomography analysis of purified human centrosomes revealed that the centre, or hub, of the cartwheel is at the end of a stalk that is linked to the side of the proximal end of the mother centriole [51] (figure 1a-1). This stalk is likely to be the physical link that keeps the procentriole and mother centriole engaged until the end of mitosis. We will follow the convention of referring to the new centriole as a procentriole while it is engaged to the mother centriole, and as a daughter centriole, once it has become disengaged.

Building the Centriole 8

Introduction

Centrioles are cylindrical structures composed of nine triplet microtubule ‘blades’ organized around a central cartwheel (Figure 1). In animal cells, centrioles can recruit microtubule-nucleating factors, called the pericentriolar material (PCM), to form a larger structure named the centrosome, which serves as the main microtubule-organizing center during both interphase and mitosis. Centrioles can also move to the cell surface and nucleate the formation of cilia and flagella: in this context, centrioles are called basal bodies (Figure 2). In recent years, genetic and functional genomic screens have identified genes essential for centriole assembly [1–11]. At the same time, proteomic analyses have identified a large list of centriole proteins, including several disease proteins, many of which remain to be further characterized [12–15]. Reflecting the fact that centrioles found in divergent eukaryotes are likely to derive from a common ancestral structure (Figure 3), and that the ultrastructural steps of centriole assembly appear to be largely conserved [16–20], many of the proteins identified in these studies are only conserved in species that assemble centrioles [13,21,22]. In addition to this conserved core of centriolar components, proteomic studies have identified a range of centriolar proteins that are unique to subsets of organisms. These differences likely reflect the different contexts in which centrioles are found, involving a variety of appendages, connecting fibers, or pericentriolar material, as well as differences in the regulation of centriole assembly (Figure 2). While great progress has been made during the past decade in understanding the molecular composition of centrioles, less is known about the assembly mechanisms that build a centriole from this large ‘parts list’. In this review, we will discuss recent work that has provided insight into the molecular mechanisms underlying the assembly of centrioles, focusing in particular on the establishment of ninefold symmetry, the control of centriole length, and the maturation of centrioles.





Figure 1
Centriole structure
Centrioles are microtubule arrays composed of nine triplets of microtubules organized around a cartwheel structure. The triplets are connected to the cartwheel through the A-tubule, the first to assemble during centriole assembly and the only complete microtubule in a triplet. The B- and C-tubules are incomplete microtubules. In vertebrates and in Chlamydomonas, the C-tubule is shorter than the A- and B-tubules and the distal end of the centriole is thus formed by doublet microtubules [112,129]. The cartwheel is formed by a central hub from which emanate spokes terminated by a pinhead structure that binds the A-tubule of the microtubule triplet. The very distal end of the centriole is decorated by ninefold symmetric distal appendages (or transition fibers) required for anchoring the centrioles at the plasma membrane when they act as a basal bodies.


Figure 2
Centrioles in centrosomes and cilia/flagella
(A) In animal cells, centrioles form the core structure of the centrosome, the main microtubule-organizing center. Quiescent cells (G0) or proliferating cells in the G1 phase of the cell cycle contain a single centrosome. The centrosome is formed by one mature centriole, the mother centriole (MC), and one non-mature centriole, the daughter centriole (DC), linked together and surrounded by a protein matrix called the pericentriolar material (PCM). In vertebrates, the mother centriole is decorated by two sets of ninefold symmetrical appendages: the distal and sub-distal appendages required for ciliogenesis and for the stable anchoring of microtubules at the centrosome, respectively. The distal appendages are conserved throughout eukaryotes, whereas the sub-distal appendages are found only in animal centrosomes. 
(B) In animals as well as in most other eukaryotes, centrioles are also required for the assembly of cilia/flagella. Centrioles, often referred to as basal bodies in this case, dock to the plasma membrane through their distal appendages and template the assembly of the nine outer microtubule doublets of the axoneme, the cytoskeletal core of cilia/flagella. A distinct structure called the transition zone separates the basal body from the axoneme. Shown are electron micrographs of (A) a human centrosome [130] and (B) the Chlamydomonas flagellar apparatus [24].


Figure 3
Conservation of the centriole and axoneme
The centriole and the axoneme, i.e. the microtubule core of cilia and flagella, are conserved features of eukaryotes. These structures were probably present in the last eukaryotic common ancestor and are still found in most branches of the eukaryotic tree of life. Centrioles and axonemes were lost concomitantly during evolution of certain taxa, most notably angiosperms and higher fungi. Taxa in which all species have lost centrioles and axonemes are indicated by a red cross. Some taxa, such as amoebozoa, comprise species that form flagella (like Physarum) as well as species completely devoid of centrioles and axonemes (likeDictyostelium). The phylogenetic tree of life is adapted from the Tree of Life web project (http://tolweb.org/tree). The last eukaryotic common ancestor (inset) is schematically represented as a single-celled organism (the plasma membrane and the nucleus are in gray) bearing two motile flagella (in black). Schematic representations of cross-sections through the centriole and the axoneme are also shown.

Centriole Functions

It seems likely that centrioles have  the primary purpose of growing cilia and flagella, which are important sensory and motile organelles found in almost all cells of the human body [23]. The ciliary microtubule doublets are continuous with the A- and B-tubules from centriole microtubule triplets (Figures 1 and ​and2)2) [24]. Proteins involved in the assembly of cilia are recruited to centrioles [25], and defects in several genes encoding centriolar proteins lead to ciliary disease phenotypes.
Despite the presence of centrioles at the mitotic spindle poles, centrioles are in many cases dispensable for mitosis, even in species that normally contain them [26]. Cell-cycle progression and cytokinesis can be defective when centrioles are missing [27–29], but this could be due to indirect effects. For example, G1 arrest in mammalian cells following centriole ablation results from an increase in stress sensitivity rather than an absolute requirement for centrioles during cell-cycle progression [30].
While centrioles are dispensable for spindle assembly, they are more important for spindle positioning. When centrioles are experimentally ablated, spindles drift within the cell [28]. In vertebrates, centriole position appears to respond to planar cell polarity cues [31], consistent with the localization of some planar cell polarity proteins at centrioles [32]. Proper spindle positioning by centrioles is thought to be necessary for proper tissue development, because defects in spindle orientation caused by mutations in centriole-associated genes can lead to nephronophthisis, a cystic kidney disease associated with abnormally wide ducts [33].

An interesting possibility is that the centriole-positioning pathway may be specific for the mother centriole, which is the oldest centriole of the two in a G1 centrosome (Figure 1) and the only fully mature centriole (i.e. with the ability to act as a basal body) in a typical vertebrate cell. This is suggested by analysis of dividing stem cells in the Drosophila male germ line, in which the mitotic spindle is always oriented such that the older centriole is anchored on the side of the cell adjacent to the stem-cell niche [34]. Similar bias in mother centriole position is seen in radial glial progenitor cells in the mouse [35]. Remarkably, following depletion of ninein, a protein required for stable anchoring of microtubules at mother centrioles, this asymmetry in mother-centriole segregation is lost, eventually leading to premature depletion of the stem-cell pool [35,36]. In Chlamydomonas mutants defective in mother–daughter cohesion, mother centrioles move to their correct position while daughter centrioles do not, suggesting the mother centriole is uniquely responsive to the positioning pathway [37].

Initiation of Centriole Assembly

Regulation of Centriole Initiation

Initiation of centriole duplication is under tight regulation to ensure the control of centriole number (for more extensive coverage of this topic, see [38–40]). In mammalian cells, a single procentriole starts forming perpendicular to the wall of each parental centriole around the G1/S transition. Once the assembly of the two new procentrioles has been initiated, further centriole duplication is inhibited until the cells pass through mitosis [41]. The release of the tight association of procentrioles with the parental centrioles, termed disengagement, occurs in late mitosis in animal cells. Disengagement involves the protease separase and Polo-like kinase 1 (Plk1) and is a prerequisite for the next round of centriole duplication [42,43]. When centrioles are absent, new centrioles can form de novo, suggesting the role of pre-existing centrioles is not actually to template the procentrioles as long proposed, but rather to bias the spatial location where the procentrioles self-assemble [44–47]. When too many centrioles are present, cells can inhibit the synthesis of new centrioles, a mechanism that allows for correction of errors in centriole number [48].

The key regulator of centriole assembly is a kinase called Polo-like kinase 4 (Plk4) or SAK in Drosophila. Inhibiting Plk4 prevents centriole duplication in both human cells and flies [49,50]. Conversely, overexpression of Plk4 and SAK can trigger the assembly of supernumerary centrioles [46,47,49,50]. Interestingly, Plk4 orthologs are not found outside the Fungi/Metazoa group (Figure 3), which suggests that centriole duplication is triggered by different mechanisms in other eukaryotes, possibly involving other Polo-like kinases [22]. Also, no Plk4 ortholog is found in Caenorhabditis elegans, in which centriole duplication is instead triggered by a non-orthologous kinase called ZYG-1 [22,51]. ZYG-1 controls the recruitment of the centriole structural component SAS-6, which is a substrate for ZYG-1 [52–54]. The recruitment of ZYG-1 itself requires SPD-2, a component of the PCM essential for centriole duplication inC. elegans embryos [7,8,52,53]. Interestingly, SPD-2 family members are only found in the genomes of Unikonts, a branch of the eukaryotic tree comprising the Fungi/Metazoa group as well as Amoebozoa, such as the model organism Dictyostelium discoideum (Figure 3) [21,22,55]. Studies of SPD-2 orthologs in human and flies suggest that the primary function of SPD-2 and related proteins is to recruit PCM around the centrioles [56–58]. A SPD-2 ortholog is also found in the matrix associated with the Dictyosteliumnuclear-associated body, a very distinctive structure that forms the core of the Dictyostelium centrosome [55]. In addition to its role in PCM recruitment, the human ortholog of SPD-2, called Cep192, is essential for centriole duplication, whereas its Drosophila ortholog appears dispensable for this process [57,58]. It is, however, possible that Cep192 affects centriole duplication indirectly through its ability to recruit PCM and microtubule-nucleating factors, as the PCM is known to play a role in centriole duplication [59]. In contrast, Drosophila Asterless (Asl) and related proteins are PCM components that appear to be more specifically required for centriole assembly [60,61]. In Drosophila, Asl localizes near the centriole wall in both proliferating cells and in testes and is required for centriole duplication in both cases [60,61]. Cep152, the vertebrate ortholog of Asl, is a component of the PCM in proliferating human cells [12]. Interestingly, zebrafish Cep152 was also found to be required for basal body assembly in multiciliated cells [61]. In these cells, up to several hundreds of basal bodies assemble at the same time around structures of unknown composition called deuterosomes as the cells undergo differentiation [62,63]. The defect observed in Cep152-depleted zebrafish supports the idea that the initiation of basal body assembly in multiciliated cells relies at least in part on the same mechanisms as centriole duplication in proliferating cells.

Establishment of the Ninefold Symmetry

Initiation of centriole assembly and establishment of the ninefold symmetry require a structure called the cartwheel (Figures 1 and ​and4).4). The cartwheel is located at the proximal end of basal bodies in a wide range of species. In vertebrate centrosomes, a cartwheel structure is present at the base of procentrioles but is no longer seen in daughter and mother centrioles [64,65]. The structure of the cartwheel has been best described in unicellular organisms. It is formed by a central hub from which emanate nine evenly spaced spokes, terminated by a pinhead structure to which microtubule triplets attach (Figure 1). InChlamydomonas, the cartwheel is assembled prior to the addition of microtubules at the tip of each spoke [18]. Two components of the cartwheel have been described in this species. CrSAS-6/Bld12p, the homolog of C. elegans SAS-6, has been proposed to be part of the inner spokes or the hub of the cartwheel [66]. Bld10p, which also belongs to a conserved protein family, has been shown to form the outer spoke and the pinhead structure [67]. Recent studies of mutants defective for these genes have provided important clues on how the cartwheel assembles and sets centriole radial symmetry. When Chlamydomonas BLD12 is deleted, most cells lack a proper centriolar structure but approximately 20% of cells assemble defective centrioles that sometimes contain an abnormal number of triplets — 7, 8 or 10 — or have missing triplets. Strikingly, the hub is missing in bld12 mutant centrioles [66]. Similarly, depletion of SAS-6 by RNA interference (RNAi) in Paramecium results in the formation of centrioles with altered numbers of triplets that retain the cartwheel spokes but are lacking the central hub [68]. A Drosophila SAS-6 null mutant is also found to have a significant reduction in the number of centrioles and forms centrioles with structural defects, for example, missing triplets [69]. As in Chlamydomonas, the Paramecium and Drosophila SAS-6 orthologs localize to the central hub of the cartwheel [68,70]. Together these results support the hypothesis that proteins of the SAS-6 family are required to build the central hub, and that the hub plays a role in establishing the ninefold symmetry [39,66,69].



Figure 4
Model for centriole assembly within duplicating human centrosomes
(A) Centriole assembly begins during late G1 or early S phase with the assembly of the cartwheel, which depends on HsSAS-6 for the central hub and Cep135 (Bld10p ortholog) for the spokes and/or the pinheads. 
(B) CPAP (SAS-4 ortholog) triggers γ-tubulin-dependent nucleation of the A-tubules and their attachment to the pinheads of the cartwheel, possibly by participating in the recruitment of the γ-tubulin ring complex (γ-TuRC) at the proximal end of the cartwheel. Each A-tubule is nucleated by a γ-TuRC and grows unidirectionally from proximal (P) to distal (D). The A-tubule remains capped by the γ-TuRC throughout the assembly process but is lost from daughter and mother centrioles. A cap structure containing CP110 and Cep97 forms at the distal end of the procentriole. The cap is required to control procentriole microtubule growth and probably also to stabilize the nascent procentriole. 
(C) The B- and C-tubules form by a γ-TuRC-independent mechanism and grow bidirectionally until they reach the length of the A-tubule. The microtubule triplets are stabilized by ε- and δ-tubulin and centriole elongation begins. 
(D) During S phase, procentrioles elongate up to ~70% of their final length. This step is dependent on hPOC5, and possibly involves hPOC1 as well. 
(E) Procentriole elongation continues after the transition into G2. Two mechanisms control centriole length at this stage: 1) a balance between the activities of CPAP, which promotes α/β-tubulin incorporation at the distal end, and the cap structure containing CP110 and Cep97; 2) an Ofd1-dependent mechanism. 
(F) After mitosis, the procentrioles become daughter centrioles. Tilted discs surrounded by electron-dense material are observed from this stage onwards in the distal part of the centriole. Markers like centrin and hPOC5 accumulate within the distal lumen as cell cycle progresses, which could reflect the progressive maturation of the centriole. HsSAS-6 is no longer associated with the proximal part of the daughter centrioles, possibly correlating with the disassembly of the central hub of the cartwheel. In contrast, the spokes could be conserved to some extent as Cep135 remains associated with mother and daughter centrioles. 
(G) After the second mitosis, centriole maturation is completed when the distal and sub-distal appendages assemble. The assembly of both types of appendage depends on ODF2, a maturation-specific marker recruited at the distal end of the daughter centriole during the previous G2 phase. Assembly of the distal appendages is also dependent on Ofd1, and possibly as well on the distal appendage component Cep164. Schematic representations of the centrosome during the successive steps of centriole assembly and maturation are shown in the lower parts of panels A–G, in which the assembling centrioles are highlighted (brown when only the cartwheel is present, yellow for later stages).

HsSAS-6, the human homolog of SAS-6, is also essential for the initial steps of centriole assembly but, unlike its homologs in other species that remain associated with mature centrioles, is no longer found associated with daughter and mother centrioles [71,72]. Loss of HsSAS-6 from the procentrioles correlates with its degradation by the 26S proteasome at the end of mitosis, and possibly also with the loss of the cartwheel structure that occurs as procentrioles become daughter centrioles (Figure 4) [64,65,71]. In contrast, SAS-6 staining is retained at the proximal end of basal bodies from rat tracheal multiciliated cells, suggesting that the cartwheel may not disassemble in this case. Intriguingly, SAS-6 also localizes to the proximal region of ciliary axonemes in these cells, revealing a possible involvement in ciliary assembly or function [73]. In C. elegans, where SAS-6 was first identified, short centrioles composed of nine singlet rather than nine triplet microtubules are formed, and no recognizable cartwheel structure is observed. Microtubule singlets are instead seen to assemble around a structure that appears as a hollow cylinder by electron microscopy. The assembly of this structure, called the central tube, requires SAS-6 [53]. Though different at the ultrastructural level, the central tube and the cartwheel thus share at least one component and are likely to be similarly required for establishing the ninefold symmetry of centrioles. Recruitment of SAS-6 within procentrioles in C. elegans requires SAS-5, a protein that physically interacts with SAS-6 and, like SAS-6, is essential for centriole duplication in this species [10,74]. Recently, a protein called Ana2 was shown to be the likely ortholog of SAS-5 in Drosophila [75]. Although poorly conserved at the amino acid level, Ana2 interacts with DSAS-6 and, like its C. elegans counterpart, is essential for centriole duplication [75–77]. Interestingly, the human ortholog of Ana2, called STIL or SIL, has been shown to be essential for proper mitotic spindle assembly and has been linked to microcephaly, reminiscent of the centriole duplication factor CPAP, the human ortholog of C. elegans SAS-4 (discussed further below) [75,78–80]. Analysis of the dynamic properties of SAS-5 in C. elegans, however, suggested that, rather than being a structural component of the centrioles, SAS-5 may be required for the recruitment of SAS-6 at procentriole assembly sites [10,74].

In addition to SAS-6-related proteins, the assembly of the cartwheel also depends on the conserved Bld10p/Cep135 family of proteins. Bld10p was originally identified as the product of a gene mutated in a Chlamydomonas strain that completely lacks basal bodies and was shown to be a component of the cartwheel spokes [9]. When the Chlamydomonas bld10 mutant is complemented by a truncated version of Bld10p, centrioles with eight triplets are often observed. These centrioles assemble around a cartwheel with shorter spokes, which seemingly leads to the formation of a centriole of smaller diameter than can only accommodate eight triplets. The cartwheel still forms nine spokes, one of which is not linked to a triplet [67]. Thus, if the central hub plays a critical role in attaining the correct ultrastructure by patterning centriole radial symmetry, the radial spokes, the length of which depends on Bld10p, do so by specifying centriole diameter. Cep135, the human ortholog of Bld10p, also localizes to the cartwheel and is essential for centriole assembly [72,81]. In contrast, Bld10/Cep135 function is apparently dispensable for the initiation of centriole assembly in Drosophila, as centrioles duplicate normally in mutant flies lacking the Bld10/Cep135 homolog [82,83]. Depletion of Drosophila Bld10 by RNAi in S2 cells leads to a partial inhibition of centriole duplication, however, suggesting Bld10 could be required in some conditions [77]. Ultrastructural analysis reveals that sperm axonemes in bld10 mutant flies contain nine outer doublet microtubules, which shows that the centrioles from which they are assembled have a proper ninefold symmetry [83]. However, sperm centrioles are shorter in these mutants than in wild-type flies and most of the flagella lack the central pair of microtubules, leading to male sterility [22,82,83]. Whether the cartwheels assembled in the bld10 mutant are normal is not known.

Steps preceding cartwheel formation remain poorly understood. In Chlamydomonas and Paramecium, the cartwheel assembles on an amorphous, disk-like structure [18]. In addition to serving as a site of cartwheel assembly, the amorphous disk could play a role in establishing the ninefold symmetry by controlling the diameter of the centrioles, and thus the number of microtubule triplets that they can accommodate. In the Chlamydomonas bld12 mutant, 70% of the basal bodies that retain a circular assembly of triplet microtubules exhibit ninefold symmetry, despite lacking the central hub of the cartwheel, suggesting additional mechanisms influence the radial symmetry of the centrioles [66].

In Paramecium, Bld10 depletion by RNAi leads to formation of centrioles that lack cartwheel spokes but retain the central hub, suggesting the hub is connected to the microtubule cylinder by another structure, most likely the amorphous disk [68]. Similar disk-like structures have not been described in animal cells: instead, procentrioles appear to be linked to the wall of the parental centrioles by a connecting stalk [65].

Conclusions

During the past decade, there has been great progress in our understanding of the biology of centrioles, yet we have only scratched the surface. The identification of major molecular players, such as SAS-6, SAS-4/CPAP, Bld10p/Cep135 and Plk4/SAK, the master regulator of centriole duplication [38,49], has allowed us to gain a much clearer picture of the initial steps of centriole assembly and how these steps are regulated, as well as the molecular bases of the ninefold symmetry. With this understanding of the upstream cues for assembly, as well the growing laundry list of centriole component proteins, we are now poised to begin to understand how centrioles are actually built, at a mechanistic level.


1) Centrosomes in Development and Disease Edited by Erich A. Nigg
2) http://www.cell.com/current-biology/fulltext/S0960-9822(06)02053-7
3) http://jcb.rupress.org/content/173/6/867.full.pdf
4) http://rstb.royalsocietypublishing.org/content/369/1650/20130460
5) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359743/
6) http://rsob.royalsocietypublishing.org/content/5/8/150082
7) http://www.cell.com/pb/assets/raw/journals/research/snapshots/PIIS0092867408016413.pdf
8  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956124/

Structural Basis of the 9-Fold Symmetry of Centrioles 1

http://www.cell.com/cell/fulltext/S0092-8674(11)00009-2

Cartwheel assembly 1

Abstract

The cartwheel is a subcentriolar structure consisting of a central hub and nine radially arranged spokes, located at the proximal end of the centriole. It appears at the initial stage of the centriole assembly process as the first ninefold symmetrical structure. The cartwheel was first described more than 50 years ago, but it is only recently that its pivotal role in establishing the ninefold symmetry of the centriole was demonstrated. Significant progress has since been made in understanding its fine structure and assembly mechanism. Most importantly, the central part of the cartwheel, from which the ninefold symmetry originates, is shown to form by self-association of nine dimers of the protein SAS-6. This finding, together with emerging data on other components of the cartwheel, has opened new avenues in centrosome biology.

Introduction
Centrioles and basal bodies (which will be referred to as centrioles unless distinction is necessary) have a characteristic structure with microtubules arranged in ninefold rotational symmetry. This structure  is strikingly well conserved among various present-day eukaryotic organisms. The precise structural conservation suggests that the nine-ness of the centriolar structure must be determined by a robust and conserved mechanism. At the centre of the mechanism is the cartwheel, a ninefold symmetrical structure that appears at the initial stage of the centriole assembly process (figure 1). It is more than 50 years ago that the cartwheel was first observed by electron microscopy [1], but only recently has great progress been made in understanding its function, molecular composition and assembly mechanism.




Figure 1.

Cartwheel as the scaffold for centriole assembly. A cartwheel assembles on an amorphous ring or disc (grey) as the first ninefold symmetrical structure appearing in the centriole assembly process. Microtubules form at the tips of the nine spokes of the cartwheel. 

2. Structure of the cartwheel

A single cartwheel is composed of a central ring (hub) from which nine filaments (spokes) emanate.

Why would and should accidental mutations or natural unguided forces produce such a highly orderd structure? 

Each spoke connects to the A-tubule of the triplet microtubule through a bulging structure called the pinhead. Usually, multiple cartwheels are stacked in the centriole lumen [1–15] (figure 1). The term ‘cartwheel’ is sometimes used to signify this entire stacked structure, but I will use the term for a single layer of this structure hereafter. While the basic structure and dimension of the hub and spoke are shared by most organisms, the number of the stacked cartwheels varies greatly among organisms and depending on the stage of centriole maturation. In most organisms, centrioles are 400–450 nm long and have several layers of stacks in the proximal approximately 100 nm of the organelle (figures 1 and 2a). However, an exceptionally long (approx. 4 μm) centriole is present in Trichonympha [1] and is shown to contain hundreds of cartwheel layers that fill approximately 90% of the lumen (figure 2d). A stage-dependent variation is found in Chlamydomonas and Spermatozopsis centrioles; centrioles in developing stages contain 7–10 layers of cartwheels, while those in the mature stage contain two to four layers (figure 2a,b) [16–18]. In mammalian centrosomes, cartwheels are present in the procentriole but disappear during mitosis (figure 2c) [8,19].




Divergence in length and position of cartwheel stacks (shown in red). 
(a) Dynamic change in the Chlamydomonas cartwheel stack during the centriole duplication cycle . As the cell cycle progresses (from left to right), the lengths of the stack and microtubules change. 
(b) The cartwheel stack inSpermatozopsis also changes in length during the cell cycle. The cartwheel stack protrudes from the centriole lumen and apparently serves as a microtubule-organizing centre [16]. 
(c) In mammals, a cartwheel stack exists in the procentriole (left), but disappears from a fully assembled centriole (right). 
(d) The Trichonympha centriole is approximately 4 μm long, while the canonical centriole is approximately 0.4 μm long. The lumen except for the distal 10% is filled with cartwheels. Thin and thick blue lines indicate triplet and singlet microtubules, respectively. (Online version in colour.)

Cryo-electron tomography determined the cartwheel structure of Trichonympha at a resolution of 38–42 Å (figure 3) [20,21]. This high resolution was achieved by averaging the images of a large number of stacked cartwheels. The reconstituted three-dimensional structure shows cartwheels occurring as stacked layers as previously thought, although there was a proposal that the extensive cartwheel stack might be produced by helical assembly of the components [22]. The central hub is a ring with a diameter of approximately 22 nm stacked with an 8.5 nm periodicity (figure 3a,b). The spoke is approximately 50 nm long between the ring and the pinhead. Interestingly, spokes in adjacent layers of cartwheel merge at approximately 20 nm from the ring, exhibiting a vertical periodicity of 17 nm, which corresponds to the size of two tubulin dimers in the microtubule protofilament (figure 3b,d). This periodicity is close to the approximately 20 nm periodicity of the cartwheel stacks observed by conventional electron microscopy in various organisms (figure 3e) [4,10,16,17]. Thus, merging of adjacent spokes, as observed in Trichonympha, may be a general feature of the cartwheel.




Figure 3.
Cryo-electron tomography images of the cartwheel in Trichonympha. 
(a) Three-dimensional representation of a stack of cartwheels (light blue) with pinheads (dark blue). Each cartwheel is composed of nine approximately 50 nm long spokes emanating from the central hub (diameter: approx. 22 nm). Scale bar, 10 nm. 
(b) Side view of the cartwheel stack highlighting the cartwheel spoke (C-SP). Hubs (left-hand side) are stacked with an 8.5 nm periodicity. Spokes in the adjacent cartwheels are paired and merged at approximately 20 nm from the hub. The spoke is structurally divided into a spoke arm (SP-A), a spoke junction (SP-J) and a spoke tip (SP-T). 
(c) Top view of the pinhead and the A-tubule of the triplet microtubule (violet). The pinhead is subdivided into a pinbody (PinB) and pinfeet (PinF) that connect the pinbody to the microtubule. Scale bar, 10 nm. 
(d) Side view of the pinhead associated with the microtubule. The pinfeet consist of pinfoot 1 (PinF1) and pinfoot 2 (PinF2), which alternate every 8 and 9 nm along the microtubule axis. The tilting of the pinfeet towards the proximal end of the centriole defines the polarity of the cartwheel. Reproduced with permission from [21]. Copyright 2013 Elsevier. 
(e) A thin section image of the Chlamydomonas centriole. The cartwheel stack displays approximately 20 nm periodicity, a distance close to that of merged spokes observed in the Trichonymphacartwheel. Scale bar, 100 nm. (Online version in colour.)


Native Architecture of the Centriole Proximal Region Reveals Features Underlying Its 9-Fold Radial Symmetry 2  

Centrioles and the related basal bodies (henceforth referred to as centrioles for simplicity) are nonmembranous organelles fundamental for the formation of cilia, flagella, and centrosomes 
 The centriole is an conserved cylindrical structure typically ∼500 nm high and ∼250 nm in diameter, which exhibits a defining 9-fold radial symmetric arrangement of peripheral microtubules. Although the question of centriole assembly has been the subject of intense investigation in recent years, a high-resolution view of the proximal region of the centriole, from where the entire structure stems, is missing.
In most species, centriolar microtubules are arranged in triplets from the proximal end through approximately two-thirds of the longitudinal axis of the centriole . These triplets comprise a complete A-microtubule oriented toward the center of the centriole, an incomplete B-microtubule attached to the wall of the A-microtubule and an incomplete C-microtubule attached to the wall of the B-microtubule . Unicellular flagellates have been instrumental in revealing the organization of microtubules along the longitudinal centriole axis. In Chlamydomonas (see Figure S1 ), the proximal-most ∼100 nm harbors microtubule triplets that are connected by a so-called A-C linker, which bridges the A-microtubule from one triplet with the C-microtubule from the neighboring one. This is followed by a ∼250 nm-long central region (also called central core), in which a Y-shaped structure is observed at the junction between the A- and B-microtubules. In the distal-most ∼150 nm, the C-microtubule is lacking, such that microtubule doublets composed of A- and B-microtubules are present.

The proximal-most ∼100 nm of the centriole contains the cartwheel, which is critical for imparting the 9-fold symmetry of the entire structure. The cartwheel consists of a central hub from which nine spokes emanate and extend toward the peripheral microtubule triplets. The  SAS-6 proteins  are essential for cartwheel assembly from unicellular organisms to human cells and are characterized by a globular N-terminal domain, followed by an extended coiled coil and a more divergent C-terminal region. SAS-6 proteins form homodimers through an interaction mediated by the coiled-coil moiety and can undergo further higher-order oligomerization through interactions between N-terminal domains of neighboring homodimers [15, 16]. These interactions involve a critical residue that, in most SAS-6 proteins, is a phenylalanine in one N-terminal domain that dives into a hydrophobic pocket in the other N-terminal domain. In vitro, such higher-order oligomerization leads to the formation of ring-like structures ∼22 nm in diameter from which emanate nine spokes [15]. Such rings are likely to exist in vivo, as revealed by the 3D map of the exceptionally long cartheel from Trichonympha [17], a symbiotic flagellate present in the hindgut of termites [18]. The Trichonympha cartwheel contains a stack of central rings spaced by 8.5 nm, which could accommodate nine homodimers of the Chlamydomonas SAS-6 protein Bld12p [17]. This analysis also uncovered that the spokes emanating from two rings merge approximately midway toward the periphery, thus forming a layer with a periodicity of 17 nm at the cartwheel margin.
Despite these advances, whether a SAS-6 protein with a canonical domain organization is present in Trichonympha is not known. Moreover, despite growing efforts to understand centriole biogenesis in a number of systems, how exactly the cartwheel is connected to the microtubule triplets is not understood. The current knowledge about this question derives from electron microscopy (EM) of resin-embedded samples, which might alter molecular organization, and does not provide sufficiently detailed information [19, 20]. Here, we use the power of cryo-electron tomography on samples captured in their native state to unveil the comprehensive 3D architecture of the proximal end of the centriole.

Figure 2

Densities Inside the Central Hub and on the Radial Spokes of the Trichonympha Cartwheel
(A) Transverse section of Trichonympha centriole tomogram. Dashed circle indicates region utilized for subtomogram averaging of the cartwheel—see (B). Scale bar represents 10 nm.
(B) Projection of reconstructed Trichonympha cartwheel. Note central hub (boxed), from which emanate nine radial spokes. Scale bar represents 10 nm.
(C) TaSAS-6(1-191) ring model fits into the 3D map of the central hub depicted with a high threshold, revealing internal densities (CID = Cartwheel Inner Densities, blue). Note that only eight heptad repeats of the TaSAS-6 coiled-coil are represented. Scale bar represents 10 nm.
(D) Magnification of the region boxed in (C), highlighting the interaction of the central hub and the CID at the TaSAS-6 N-N interface. Yellow spheres indicate Tyr118. Scale bar represents 2 nm.
(E) 3D representation depicted with a low threshold in order to clearly visualize the three layers of the cartwheel (light blue), each with two TaSAS-6 rings, plus a part of the peripheral Pinhead (dark blue). Note that at this lower threshold density, the CID appears as a continuous disk. Scale bar represents 10 nm.
(F) Longitudinal view of the cartwheel highlighting the radials spokes (C-SP), which can be divided into three parts: the spoke arm (SP-A), the spoke junction (SP-J), and the spoke tip (SP-T); part of the Pinhead is also visible (dark blue).

Figure 3

The Pinhead Can Be Subdivided into a Pinbody and Two Pinfeet and Is Polarized along the Proximal-Distal Centriole Axis
(A) Transverse section of Trichonympha centriole tomogram. Dashed circle indicates region utilized for the subtomogram averaging in the reconstructions of Figures 3, 4, and 5. Scale bar represents 10 nm.
(B) Projection of reconstructed microtubule triplet. The radial spoke is connected with the A-microtubule through the Pinhead and the A-microtubule is attached to the C-microtubule of the neighboring triplet via the A-C linker. Scale bar represents 10 nm.
(C) 3D map of the region boxed in (B), showing the A-microtubule with the Pinhead. The entire Pinhead is 15 nm long and interacts with protofilament A3 of the A-microtubule; the Pinhead can be subdivided into the Pinbody (PinB) and the Pinfeet (PinF). Note microtubule inner protein (MIP, red arrow) visible next to protofilament A9. Scale bar represents 10 nm. The yellow, red, and blue arrows correspond to the X, Y, and Z axes, respectively.
(D and E) Side views of the Pinhead highlighting the Pinfeet (PinF1 and PinF2), which alternate every 8 and 9 nm. Note also polarity along the proximal-distal centriole axis marked by the asymmetric arrangement of the Pinfeet, as well as the 50° angle between the Pinfoot and the Pinbody.

Figure 4

Architecture of the Microtubule Triplet in the Proximal Region of the Centriole
(A) 3D representation of microtubule triplet with protofilament numbers in A-, B-, and C-microtubules. Scale bar represents 10 nm.
(B) Inside view of microtubule triplet revealing the 8.5 nm periodicity of the B10 junction between the A- and B-microtubules, as well as of the C10 junction between the B- and C-microtubules.
(C) Outside view of microtubule triplet highlighting the C-microtubule with the C-stretch (extending from the C1 protofilament) exhibiting an 8.5 nm periodicity.

Observe how the periodicity of the A- and B-microtubules precisely fit the PinF1 and PinF2 periodicity of the pinhead. These are two distinct parts, namely the pinhead of the wheel, and the microtubules, and their  inverval of the A- and B-microtubules which  fit precisely togheter. Unless there is a distant goal in mind, there is no way that natural mechanisms would produce these precisely, machine like structures and parts by trial and error. Upon our experience we know, however, that creative minds do invent and produce machines and machine parts that fit together with a goal of creating functional machines. 

Figure 5

The A-C Linker Can Be Subdivided into the A-Link and the C-Link and Exhibits Polarity along the Proximal-Distal Centriole Axis
(A) Top view of two microtubule triplets connected through the A-C linker (box), which links the A8-protofilament and the C9-protofilament. Scale bar represents 10 nm.
(B) External side view of the A-C linker revealing two densities, the A-link and the C-link. The A-link exhibits a ∼60° angle inclination from the microtubule protofilament axis. Note also the 8.5 nm periodicity along the A- and C-microtubules.

Figure 6

Cartwheel and Intertriplets Connections Are Conserved between Trichonympha and Chlamydomonas Proximal Regions of Centrioles
(A) Top view of merged 3D reconstructed maps of the cartwheel (blue), the Pinhead (dark blue), and the microtubule triplet (purple). Note that a similar threshold has been used to fit the Pinhead from the cartwheel map into the Pinhead of the microtubule triplet map.

Figure 7

Complete 3D Architecture of the Proximal Region of the Centriole
Overall 3D map of the procentriole (when viewed from the proximal end) highlighting the different structures analyzed in this study. Scale bar represents 20 nm.

Another important finding from the tomography study is that the pinhead has a complex structure polarized in the direction of the centriole axis [21]. The pinhead consists of a hook-like protrusion (pinbody) and two linkers (pinfeet) present between the pinbody and the microtubule (figure 3c,d). The polarity is defined by a tilt of pinfeet towards the proximal end of the centriole (figure 3d). The presence of polarity is interesting because the central part of the cartwheel is composed of nine SAS-6 dimers (see §4a) and the dimer has a twofold symmetry with respect to the axis of the coiled-coil spoke; in other words, the core structure of the cartwheel has no polarity in the direction of the centriole axis [23,24].


Regarding the structural polarity of the cartwheel stack, interesting observations have been made in some bryophyte species [13] and a marine protist, Labyrinthula [14], which may provide an insight into the process of microtubule assembly on the cartwheel. The process of de novo centriole formation, which occurs during mitosis in these organisms, suggests that two new centrioles assemble bidirectionally from a preassembled cartwheel stack, and the resulting centrioles share the stack at their proximal ends (figure 4). Although it is possible that these centrioles (a bicentriole) are formed by joining of two mature centrioles through their proximal ends, like the colinearly arranged mother and daughter centrioles observed in quiescent mammalian cells [25], morphological markers of mitotic stages strongly suggests that, in these organisms, the bicentriole forms first and thereafter its binary fission produces two mature centrioles. Given that the cartwheel polarity resides only in the pinhead, and that the pinhead is composed of a component(s) different from the rest of the cartwheel as I will discuss later, it seems possible that the cartwheel stack may first assemble without pinheads and polarity, and pinheads and microtubules assemble on the cartwheel afterwards.





1) http://rstb.royalsocietypublishing.org/content/369/1650/20130458
2) http://www.cell.com/current-biology/fulltext/S0960-9822(13)00786-0

The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies  1

Abstract

Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

Most animal cells contain structures known as centrioles. Typically, a cell that is not dividing contains a pair of centrioles. But when a cell prepares to divide, the centrioles are duplicated. The two pairs of centrioles then organize the scaffolding that shares the genetic material equally between the newly formed cells at cell division.

Centriole assembly is tightly regulated and abnormalities in this process can lead to developmental defects and cancer. Centrioles likely contain several hundred proteins, but only a few of these are strictly needed for centriole assembly. New centrioles usually assemble from a cartwheel-like arrangement of proteins, which includes a protein called SAS-6. Previous work has suggested that in the fruit fly Drosophila melanogaster, Sas-6 can only form this cartwheel when another protein called Ana2 is also present, but the details of this process are unclear.

Now, Cottee, Muschalik et al. have investigated potential features in the Ana2 protein that might be important for centriole assembly. These experiments revealed that a region in the Ana2 protein, called the ‘central coiled-coil domain’, is required to target Ana2 to centrioles. Furthermore, purified coiled-coil domains were found to bind together in groups of four (called tetramers). Cottee, Muschalik et al. then used a technique called X-ray crystallography to work out the three-dimensional structure of one of these tetramers and part of the Sas-6 protein with a high level of detail. These structures confirmed that Sas-6 proteins also associate with each other.

When fruit flies were engineered to produce either Ana2 or Sas-6 proteins that cannot self-associate, the flies' cells were unable to efficiently make centrioles. Furthermore, an independent study by Rogala et al. found similar results for a protein that is related to Ana2: a protein called SAS-5 from the microscopic worm Caenorhabditis elegans.

Further work is needed to understand how Sas-6 and Ana2 work with each other to form the cartwheel-like arrangement at the core of centrioles.

Introduction

Centrioles are complex microtubule (MT) based structures that are required for the formation of centrosomes and cilia/flagella. These organelles have many important functions in cells, and their dysfunction has been linked to a plethora of human pathologies, ranging from cancer to microcephaly to obesity (Nigg and Raff, 2009; Bettencourt-Dias et al., 2011). Thus, understanding how these organelles assemble and function is an important goal of both basic and biomedical research.
Although several hundred proteins are thought to be concentrated at centrioles, only a small number appear to form a conserved ‘core’ pathway that is essential for centriole assembly (Delattre et al., 2006; Pelletier et al., 2006; Gönczy, 2012). During canonical centriole duplication, the protein kinase Plk4/Sak/ZYG-1 is recruited to the mother centriole by SPD-2 in worms (Delattre et al., 2006;Pelletier et al., 2006; Shimanovskaya et al., 2014), by Asterless (Asl) in flies (Blachon et al., 2008;Dzhindzhev et al., 2010), or by a combination of the two (Cep192 and Cep152, respectively) in humans (Cizmecioglu et al., 2010; Hatch et al., 2010; Kim et al., 2013; Sonnen et al., 2013). The protein kinase recruits STIL/Ana2/SAS-5 and Sas-6 to a single site on the side of the mother centriole where they assemble with CPAP/Sas-4 into a cartwheel structure that helps to establish the ninefold symmetry of the centriole (Dammermann et al., 2004; Delattre et al., 2004; Leidel et al., 2005; Nakazawa et al., 2007; Peel et al., 2007; Strnad et al., 2007; Stevens et al., 2010a; Tang et al., 2011; Arquint et al., 2012). CPAP/Sas-4 can interact with tubulin (Hung et al., 2004) and is required to recruit the centriole MTs to the outer region of the cartwheel (Pelletier et al., 2006), possibly working together with Cep135/Bld10 (Hiraki et al., 2007; Lin et al., 2013)—although no homologue of this protein has been identified in worms, and it does not appear to be essential for centriole duplication in flies (Carvalho-Santos et al., 2012; Mottier-Pavie and Megraw, 2009; Roque et al., 2012).

Great progress has been made recently in understanding how these proteins interact and how these interactions are regulated to ensure that a new centriole is only formed at the right place and at the right time. In particular, the crystal structure of Sas-6 from several species has revealed how this protein forms a dimer through its C-terminal coiled-coil domain (C–C) that can then further homo-oligomerise through an N-terminal headgroup interaction (N–N) to form a ring structure from which the C–C domains emanate as spokes (Kitagawa et al., 2011; van Breugel et al., 2011, 2014; Hilbert et al., 2013). This Sas-6 ring structure can be modelled into EM tomographic reconstructions of the cartwheel from Trichonympha centrioles (Guichard et al., 2012, 2013), strongly suggesting that these Sas-6 rings form the basic building blocks of the cartwheel. In support of this hypothesis, mutant forms of Sas-6 that cannot homo-oligomerise through the N–N interaction are unable to support efficient centriole duplication (Kitagawa et al., 2011; van Breugel et al., 2011), although they can still target to centrioles, a function that seems to rely on the C–C domain (Fong et al., 2014; Keller et al., 2014).

A crystal structure of the interface between Ana2/STIL and Sas-4/CPAP has also recently been solved (Cottee et al., 2013; Hatzopoulos et al., 2013), as has the interaction interface between Plk4 and both Cep192/SPD-2 and Cep152/Asl (Park et al., 2014); mutations that perturb these interactions in vitro perturb centriole duplication in vivo, indicating that these interactions are also essential for centriole duplication. More recently, it has been shown that Plk4 can recruit STIL to centrioles in human cells (Ohta et al., 2014; Kratz et al., 2015) and that Plk4/Sak can phosphorylate the conserved STIL/Ana2 (STAN) domain in STIL/Ana2 proteins in humans and flies, thereby promoting the interaction of the STAN domain with Sas-6 (Dzhindzhev et al., 2014; Ohta et al., 2014;Kratz et al., 2015). Mutant forms of STIL/Ana2 that could not be phosphorylated strongly perturbed Sas-6 recruitment to centrioles and centriole duplication. Together, these studies have shed important light on the molecular mechanisms of centriole assembly, but many important questions remain.

In particular, it has been proposed that the homo-oligomerisation properties of Sas-6 establish the ninefold symmetry of the centriole (Kitagawa et al., 2011), and, remarkably, a ninefold symmetric ring structure is formed in crystallo by Leishmania major Sas-6 (van Breugel et al., 2014). However, although Sas-6 oligomers appear to have a propensity towards ninefold symmetry, Sas-6 proteins spontaneously assemble into oligomers of varying stoichiometry in vitro (Kitagawa et al., 2011; van Breugel et al., 2011), suggesting that the homo-oligomerisation properties of Sas-6 alone may be insufficient to enforce the rigorous ninefold symmetry that is observed in centrioles from virtually all species (Cottee et al., 2011). Additionally, recent Cryo-EM analysis suggests that the basic building block of the cartwheel stack is not a single ring and spoke structure, but rather a pair of rings that sit on top of one another: these rings do not make direct contact with each other, but are joined in the more peripheral regions through their spokes (Guichard et al., 2012, 2013). Our current knowledge of Sas-6 self-association cannot explain this important feature of the cartwheel structure.


We previously showed that overexpressed Sas-6 can form higher-order aggregates in Drosophilaspermatocytes, but these aggregates only adopt a cartwheel-like structure when Ana2 is also overexpressed (Stevens et al., 2010b), and the STIL/Ana2 protein family is essential for the proper recruitment of Sas-6 to centrioles (Dzhindzhev et al., 2014; Ohta et al., 2014). We reasoned therefore, that Ana2 was likely to also play an important part in determining the structure of the central cartwheel. We set out to investigate the potential structural features of Ana2 that might be important for centriole assembly.

The CCCD is required for the centriolar targeting of Ana2

The Drosophila Ana2 protein contains four regions that have significant homology to Ana2/STIL proteins from other species (Cottee et al., 2013). Fly Ana2 lacks the conserved region 1 found towards the N-terminus in vertebrate STIL proteins (Figure 2A), but contains a CR2 domain that interacts with Sas-4 (Cottee et al., 2013; Hatzopoulos et al., 2013), a predicted central coiled-coiled domain (CCCD), a STAN domain (Stevens et al., 2010a) that interacts with Sas-6 (Dzhindzhev et al., 2014; Ohta et al., 2014) and a short C-terminal CR4 domain  (Cottee et al., 2013). To examine the potential function of these conserved regions, we synthesised mRNAs in vitro that contained either wild type (WT) or truncated versions of Ana2 fused to either an N- or C-terminal GFP . These mRNAs were injected into WT early embryos (that contain unlabelled endogenous WT Ana2 protein) expressing RFP-Centrosomin (Cnn) as a centrosomal marker (Conduit et al., 2010). The localisation of the encoded GFP-fusion protein was assessed 90–120 min after mRNA injection .

The CCCD forms a stable tetramer in solution

We reasoned that the CCCD might function as an oligomerisation domain for Ana2 ( oligomers composed of two, three and four monomers )    . To test this possibility, we bacterially expressed and purified the 37aa CCCD region (residues 193–229)—as predicted by the COILS server (Lupas et al., 1991)—as a His-tagged diLipoyl peptide  (Cottee et al., 2013). A SEC-MALS analysis revealed that the purified protein, either with or without the Lipoyl tags, formed a tetramer at a wide range of concentrations (36–900 μM) . The CCCD tetramer was very stable and we could not find in-solution conditions under which it was dissociated, so we could not calculate a Kd. Even when examined using the usually denaturing technique, Electrospray-Ionisation Mass Spectrometry, the tetramer did not fully disassemble . We also expressed and purified the 42aa predicted CCCD (residues 717–758) from the human STIL protein as a His-tagged diLipoyl peptide. This also formed a tetramer, although this was less stable than the fly CCCD tetramer and only formed at higher protein concentrations .

Crystal structure of the Ana2 CCCD

The purified Ana2 CCCD protein readily formed protein crystals that diffracted extremely well, enabling us to refine a structure to 0.80 Å resolution (Figure 3, Figure 3—figure supplement 1, Table 1). The structure demonstrated that the Ana2 CCCD forms a parallel, symmetrical 4-helix bundle, with a left-handed supercoil (Figure 3A). This structure appears to be unusual as we could find only one other natural soluble protein in the PDB that homo-tetramerises through a parallel four-helical bundle (NSP4, a tetrameric enterotoxin secreted by rotaviruses). Analysis using the PISA server (Krissinel and Henrick, 2007) showed that residues located at the g, a, d and e positions of the helical heptad repeat were all buried at the tetramer interface (Figure 3A, yellow residues). The tetramer is stabilised by at least three mechanisms: first, the knob-into-holes and van der Waals packing of hydrophobic residues (Figure 3B,C); second, the packing of internally facing polar residues (Figure 3D); third, a cross-chain salt bridge formed between R208 and E210 (Figure 3E).



The Ana2 CCCD forms a parallel four helical tetramer.
(A) Left, the structure of the Ana2 CCCD tetramer generated around the crystallographic fourfold symmetry axis. The primary amino acid sequence is shown above the structure; residues in the g, a, d and e positions of the helical heptad repeat are indicated below the sequence. All these residues were ≥30% buried (according to PISA server analysis) and are coloured in yellow, with side-chains in stick format—other residues are coloured in cyan (side-chains not shown). The TEV cleavage remnant is shown in grey. Right, schematic transverse view of the tetramer indicating how the g, a, d and e residues of the heptad repeat are buried at the tetramer interface. Note that the g and d residues (coloured red, and highlighted with a red circle underneath the primary amino acid sequence) form one side of this interface; these 10 residues were mutated to generate forms of the protein that could no longer form tetramers (see main text). 
(B–E) Schematics illustrate the molecular determinants of tetramerisation, with interfacing residues shown as grey sticks. 
(B) A hydrophobic cluster of interface residues. The labelled residues sit at the g, a, d and e positions of the heptad repeat, and pack closely forming a hydrophobic environment. 
(C) A side on view of the same cluster, with one chain shown as a surface. 
(D) A transverse N-C view of a QQQ triad which adopts positions g, a and b of the heptad. These polar side-chains form an inward facing hydrogen-bond network. 
(E) A side-on view showing a salt bridge between adjacent chains of the tetramer.

Mutations that perturb Ana2 tetramerisation in vitro perturb centriole duplication in vivo

To test the potential importance of tetramerisation of the CCCD in vivo, we created point mutations within the CCCD that our structural studies suggested would disrupt the ability of the CCCD to tetramerise. We replaced all ten residues at the d and g positions of the CCCD with either Ala (CCCD-A), Ser (CCCD-S) or Asp (CCCD-D) (Figure 3A, residues circled in red). A SEC-MALS analysis revealed that all of these mutant CCCD proteins behaved as monomers rather than tetramers in vitro (Figure 4A). We then made equivalent CCCD mutations within the context of the full length Ana2 protein and tested their localisation in our embryo RNA injection assay. All three mutant proteins were undetectable at centrioles but still localised diffusely to the PCM (Figure 4B–D), indicating that the mutant proteins are not simply misfolded or degraded, as the STAN domain can still target them to the PCM.

The ability of Ana2 to tetramerise is important for Ana2 function and for centriole assembly, but that Ana2-CCA retains some residual ability to promote the assembly of CLSs in vivo.

Drosophila Sas-6 can homo-oligomerise to form a canonical cartwheel structure

It has previously been shown that Sas-6 proteins also need to homo-oligomerise to function in centriole duplication (Kitagawa et al., 2011; van Breugel et al., 2011), so we wanted to explore the relative importance of Sas-6 and Ana2 oligomerisation for centriole duplication. In all species examined to date Sas-6 forms dimers through an extended C-terminal coiled-coil region (C–C) (Kitagawa et al., 2011; van Breugel et al., 2011; Qiao et al., 2012). In Danio rerio, Chlamydomonas and Leishmania these dimers can further homo-oligomerise through an N-terminal headgroup interaction (N–N) to form a flat ninefold symmetric ring from which the C–C domains emanate—thus forming the central hub and spokes of the cartwheel (Figure 6I). In Caenorhabditis elegans, however, the SAS-6 headgroup-CC orientation is altered (Figure 6H), and SAS-6 dimers appear to oligomerise into a spiral, rather than a flat-ring (Hilbert et al., 2013), potentially explaining why a classical cartwheel with nine spokes has not been visualised by EM inC. elegans centrioles (Pelletier et al., 2006). In Drosophila centrioles, EM images reveal a clear central cartwheel hub from which emanating spokes are often visible—but it is difficult to visualise more than a few spoke structures at any one time (e.g., Callaini et al., 1997; Roque et al., 2012; Helio Roque, personal communication), making it unclear whether Drosophila Sas-6 oligomerises into a canonical ring or into a spiral. To address this issue, we attempted to examine the structure of Drosophila Sas-6 (Figure 6A).




Figure 6.
A biochemical and structural analysis of Drosophila Sas-6.
(A) A schematic representation of Drosophila Sas-6 highlighting the position of the N-terminal head domain (blue) and C-terminal coiled-coil (CC) domain (green). Red lines below represent the constructs used in SEC-MALS and EM studies (top) and in X-Ray Crystallography studies (bottom). 
(B) A SEC-MALS analysis of WT (blue trace) and F143D mutant (red trace) Sas-61–241 proteins, injected at 33 µM. The horizontal black line and grey bar represent the theoretical dimer mass ±5% tolerance. The WT protein could not be analysed by MALS as it eluted in the void volume and appeared to form a range of higher-order oligomers. 
(C) Negative-stain EM analysis of purified WT ([i]–[iv]) Sas-61–241 protein, showing the chain-like structures formed ([iii] and [iv] show magnified views of the red boxed areas in [i] and [ii]); these structures are not detectable in preparations of the mutant Sas-6-F143D1–241 protein ([v]). 
(D) The structure of the Sas-6 dimer, coloured according to Consurf conservation scores (Glaser et al., 2003) from cyan(variable) to burgundy (conserved). The conserved PISA domain and the N-CC interface regions are highlighted with dashed circles. 
(E–G) Superimposed structures from D. melanogaster, D. rerio,Chlamydomonas and Leishmania (as indicated) of the Sas-6 N-terminal head-group with a short stretch of the coiled-coil domain. 
(H) Superimposed structures of the N-CC interface in D. melanogaster, D. rerio,Chlamydomonas and C. elegans. Note how the interface is rotated by ∼30° in C. elegans (purple) compared to the other structures. 
(I) The DmSas-6 structure modelled into a ninefold symmetric flat ring (green, single dimer shown in red), similar to that observed in crystallo for LmSAS-6. This ring structure was docked into the EM density of the Triconympha cartwheel structure (Guichard et al., 2013) (cyan surface, cut away to reveal the DmSAS-6 ring).

We were unable to purify constructs containing only the N-terminal head-group, however we could purify constructs that contained the N-terminal headgroup and either 59 (Sas-61–216) or 84 (Sas-61–241) residues of the predicted C–C region. In initial attempts to purify Sas-61–241 the protein invariably formed large aggregates (blue trace, Figure 6B) that appeared to be elongated chains of protein by negative-stain EM (Figure 6Ci–iv). It has previously been shown that a large hydrophobic residue in the headgroup is essential for the N–N interaction in several species (Kitagawa et al., 2011; van Breugel et al., 2011), so we mutated the equivalent residue, F143, to Asp. Purified Sas-61–241-F143D behaved as a dimer by SEC-MALS (red trace, Figure 6B) and aggregates were no longer detectable by negative-stain EM (Figure 6Cv); we conclude that aggregate formation is dependent upon the N–N interaction, and the F143D mutation perturbs this interaction in vitro.
To investigate how Drosophila Sas-6 might oligomerise into a cartwheel we solved the crystal structure of Sas-61–216-F143D to 2.9 Å (Figure 6D, Table 2). The asymmetric unit contained a dimer of Sas-6, associated via the coiled-coil interface. To assess whether this Sas-6 N-CC dimer could be built into a canonical flat ring structure, we compared it to other Sas-6 orthologues for which structures are available. The DmSas-6 N-CC dimer could be superimposed with Sas-6 N-CC dimers from D. rerio,Chlamydomonas and Leishmania Sas-6 (average pairwise RMSD 1.87 ± 0.31 Å over 617 ± 47 backbone atom pairs) (Figure 6E–G). However it could not be superimposed onto C. elegans SAS-6, which has an alternative head-group-spoke conformation (Figure 6H). Furthermore, we found that the DmSas-6 N-CC dimer could be modelled into a flat ninefold ring (Figure 6I), similar to that observed in crystallo forLeishmania Sas-6 (van Breugel et al., 2014). The structure of DmSas-6 is therefore highly similar to Sas-6 orthologues from organisms with canonical cartwheels, suggesting that it also forms such a structure.

Mutations that perturb Sas-6 oligomerisation in vitro perturb centriole duplication in vivo

To test whether the ability of Sas-6 to form higher-order oligomers was important for Sas-6 function, as has been observed in several other systems (Kitagawa et al., 2011; van Breugel et al., 2011), we generated stable transgenic lines expressing either WT GFP-Sas-6 or GFP-Sas-6-F143D under the control of the ubiquitin promoter. This promoter consistently resulted in the overexpression of both WT GFP-Sas-6 and GFP-Sas-6-F143D compared to the endogenous protein (Figure 7A). While WT GFP-Sas-6 strongly rescued the centriole duplication defect seen in Sas-6 mutants, GFP-Sas-6-F143D rescued much more weakly, although, at least one CLS was detectable in ∼60% of cells expressing one copy of the transgene (Figure 7B–F′′). As was the case with the rescue of the ana2 mutation by Ana2-CCA-GFP, these structures stained for multiple centriole/centrosome markers and were usually located at the spindle poles in mitotic cells, demonstrating that they retain at least some centriole and centrosome function (Figure 7F–F′′; data not shown). From our qualitative analysis, however, the CLSs formed when Sas-6 mutants were rescued by GFP-Sas-6-F143D often appeared smaller and more fragmented than those observed when ana2 mutants were rescued by Ana2-CCA-GFP, suggesting that the CLSs formed in the presence of GFP-Sas-6-F143D may be less well organised than those formed in the presence of Ana2-CCA-GFP. Moreover, as described below, females carrying even one copy of this transgene invariably laid embryos that arrested early in development, so we could not generate flies carrying two copies of the transgene to test if the rescuing activity of the transgene increased with gene dosage—as we observed for Ana2-CCA-GFP (Figure 5B). Nevertheless, these data demonstrate that the ability of Sas-6 to form higher order oligomers is important for Sas-6 function and for centriole assembly, but that GFP-Sas-6-F143D retains some residual ability to promote the assembly of CLSs in vivo (Figure 6B,C).

Discussion

It is now widely accepted that the structure of the centriole cartwheel is formed around a core of 9 Sas-6 dimers that homo-oligomerise to form a ring structure (Cottee et al., 2011).


SAS-6 oligomerization: the key to the centriole? 2

Centrioles are among the most beautiful of biological structures. How their highly conserved nine-fold symmetry is generated is a question that has intrigued cell biologists for decades. Two recent structural studies provide the tantalizing suggestion that the self-organizing properties of the SAS-6 protein hold the answer.






 Sas-6 molecules can form such ninefold symmetric rings in the absence of any other proteins in vitro, and mutations that perturb the ability of Sas-6 to homo-oligomerise in vitro strongly perturb centriole assembly in vivo (Kitagawa et al., 2011;van Breugel et al., 2011, 2014). We previously showed, however, that overexpressed Sas-6 can only form cartwheel-like structures in fly spermatocytes when Ana2 is also overexpressed (Stevens et al., 2010b). Here we show that Ana2/STIL proteins also homo-oligomerise and that mutations that perturb the homo-oligomerisation of fly Ana2 in vitro also strongly perturb centriole assembly in vivo. Thus, Sas-6 homo-oligomerisation alone appears unable to drive efficient cartwheel assembly in vivo if Ana2 is unable to homo-oligomerise.

Our initial structure/function analysis of Ana2 revealed that the CCCD is important for the recruitment of Ana2 to centrioles. This is consistent with the recent discovery that the CCCD in human STIL interacts with Plk4 and is important for STIL recruitment to centrioles (Ohta et al., 2014; Kratz et al., 2015). In both flies and vertebrates, Sak/Plk4 can also phosphorylate the STAN domain of Ana2/STIL, promoting its interaction with Sas-6, and allowing it to recruit Sas-6 to the newly forming centriole (Dzhindzhev et al., 2014; Ohta et al., 2014; Kratz et al., 2015). Interestingly, we found that although the STAN domain could not localise Ana2 to centrioles in fly embryos in the absence of the CCCD, the centriolar localisation of Ana2 was much weaker in the absence of the STAN domain, suggesting that an interaction with Sas-6 is required for robust Ana2 localisation in flies. This result is in agreement with the finding that fragments of STIL containing both the CCCD and the STAN domain interact most strongly with Plk4 (Kratz et al., 2015), but contrasts with reports in human cells where deleting the STAN domain did not affect STIL localisation to centrioles (Ohta et al., 2014) and in fly cultured cells, where the depletion of Sas-6 by RNAi did not detectably perturb the centriole localisation of Ana2 (Dzhindzhev et al., 2014). These latter results suggest that the proper recruitment of Ana2/STIL to centrioles is independent of the STAN domain's interaction with Sas-6. Our findings suggest, however, that although Plk4 may initially recruit some Ana2 to centrioles without Sas-6, the subsequent incorporation of Ana2 into the new centriole is dependent upon the successful assembly of a cartwheel structure, and this cannot occur without Sas-6. In addition, although Plk4 can clearly recruit Ana2/STIL to centrioles in flies and humans, in worms ZYG-1 (the Plk4 functional homologue) can directly recruit SAS-6 (Lettman et al., 2013). Thus, the molecular detail of the interactions between Plk4/Sak/ZYG-1, Sas-6 and Ana2/STIL/SAS-5 involved in cartwheel assembly remain to be fully elucidated, and may vary between different cell types and species.

Molecular mechanism of cartwheel assembly

(a) Cartwheel central part assembled from SAS-6/Bld12p

SAS-6/Bld12p is a component of the central part of the cartwheel. What mechanism assembles this protein into the ninefold symmetrical structure? The answer came from X-ray crystallography and biochemical analyses performed independently by two groups. The two groups used SAS-6/Bld12p of three different organisms, C. elegans, Chlamydomonas and zebrafish, but the results obtained are similar to each other, indicating the conserved function of this protein. SAS-6/Bld12p consists of a conserved N-terminal domain, a coiled-coil domain and a non-conserved C-terminal domain. Crystallography and biochemical analyses of a fragment containing the N-terminal domain attached with part of the coiled-coil domain, and another fragment containing the N-terminal domain alone, revealed that this protein forms a stable homodimer via association of the coiled-coil domains to form a long coiled-coil tail (figure 6a), and that the dimers interact with each other through a hydrophobic interaction between the head domains (figure 6b). In the latter interaction, an Ile (C. elegans) or a Phe (Chlamydomonas and zebrafish) residue within a loop in one head is inserted into a hydrophobic pocket in the other head. Although this hydrophobic interaction is weak (Kd for C. elegans, zebrafish and human SAS-6s are approx. 110, approx. 90 and approx. 50 μM, respectively), mutational analyses demonstrated that it is essential for centriole assembly. These results led to a model in which nine dimers assemble into a ring through the head-to-head interaction, and the head ring and the coiled-coil tails constitute the hub and the spokes of the native cartwheel (figure 6c). In fact, SAS-6 dimers in solution form oligomers, including nonamers [24], and a ring structure having a diameter close to that of the cartwheel hub [23]. The model predicts that the C-terminus of the SAS-6 molecule should be localized distal to the hub. Immunoelectron microscopy of Chlamydomonas cells expressing Bld12p tagged with HA at the C-terminus confirms the predicted orientation [24,51]. Considering the cartwheel function in centriole assembly, all these findings collectively indicate that the oligomerization properties of SAS-6/Bld12p are crucial for stabilization of the ninefold symmetry of the centriole.


1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471874/
2) http://www.nature.com/nchembio/journal/v7/n10/full/nchembio.660.html
3) http://rstb.royalsocietypublishing.org/content/369/1650/20130458

Plk4-dependent phosphorylation of STIL is required for centriole duplication 1

Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL) and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process.

Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication.

How could and why would natural, unguided mechanisms and processes emerge with the "drive" or necessity to 1. produce STIL and Sas-6 proteins, and 2. make them accumulate at the initial stage of the centriole assembly process, at the right place , at the right time, and the right quantity, and trigger phosphorilation required to duplicate centrioles ? 


1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359743/

The centriole duplication cycle 1

 What defines the location of the origin of centriole duplication? Emerging evidence suggests that three centriole proteins, PLK4, SASS6 and STIL, have an instructive function in this process as they all localize at the site of procentriole assembly at the G1–S transition, when centriole assembly is initiated. SASS6 is a structural component of the cartwheel, the ninefold symmetric template structure internal to the proximal end of the centriole [28–30], and STIL is associated with SASS6 [31–34].
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 In Caenorhabditis elegans, SAS-6 is recruited to the centriole by ZYG-1, a functional orthologue of PLK4, thus this might be an evolutionarily conserved mechanism of defining the site of centriole assembly [38]. Strikingly, PLK4 at the centriole changes from a ring-like localization around the centriole barrel (early G1) to the dot-like localization (G1–S transition), and this change coincides with initiation of downstream events of centriole duplication, further supporting the role of PLK4 in marking the origin of centriole duplication [36]. These studies identify PLK4 as the earliest marker that localizes to the site of procentriole assembly, and given that PLK4 is a protein kinase, the most straightforward model would be that PLK4 phosphorylation of downstream proteins in centriole assembly initiates the process. Although some proteins have been shown to be substrates of PLK4 in vitro, including some centriole duplication proteins [39–42], there is as yet no direct link between PLK4 kinase activity and centriole initiation.

If PLK4 defines the origin of centriole duplication, what targets PLK4 to the centrioles at the right time and place? Recent studies suggest that two PLK4-binding proteins are important in this process. PLK4 (or ZYG-1) recruitment to the centrioles depends on Asterless in Drosophila [43] and on SPD-2 in C. elegans[44,45]. Interestingly, in mammalian cells, the orthologues of these two proteins, CEP152 and CEP192, respectively, interact with PLK4 and cooperate in the recruitment of PLK4 to the centrioles [36,46] (figure 1a-1). This recruitment depends on electrostatic interactions between the positively charged polo-box domain of PLK4 and acidic regions of CEP192 and CEP152 [36,46]. Both CEP152 and CEP192 are distributed symmetrically around the circumference of the centriole barrel [37,47], thus, although these studies provide an appealing explanation for how PLK4 is recruited to centrioles, they do not address the fundamental question, which is how is a single site for initiation of a morphological event chosen from a radially symmetric surface? This is, in essence, a symmetry-breaking event, similar to that in choosing a single site for bud formation in budding yeast [48], or establishment of the axes of a C. elegans embryo [49,50]. In these cases, and presumably in centriole initiation, there is some form of cooperativity or positive feedback that results in asymmetric accumulation of the relevant proteins in a symmetric background. It is possible, then, that association of PLK4 with CEP192 and/or CEP152 has such cooperative properties, or that the kinase activity of PLK4 provides some positive feedback mechanism to that association. Importantly, such a model would depend critically on the concentration of PLK4 in the cell, and that the concentration be subsaturating with respect to its binding partners; as described below, the concentration of PLK4 is known to be critical to limiting duplication to one site.

How are microtubules added to the ninefold symmetric cartwheel to form the microtubule triplets found in most species? gamma-tubulin, in association with the cartwheel, nucleates the A-tubule, which then grows as the centriole elongates. Following the nucleation and elongation of the A-tubules, the B- and C- tubules form, but are not capped at their minus end, suggesting that their assembly is initiated by a different mechanism (figure 1a-3).

We propose the following as a general model for centriole duplication control in animal cells.

• (i) At the G1/S transition, the mother centriole acquires a single focus of PLK4 by a cooperative binding/positive feedback mechanism, creating an origin of duplication that initiates procentriole formation.
  
  
• (ii) The mother centriole does not initiate a second procentriole, because PLK4 is limiting and cooperativity ensures that all free PLK4 goes to the existing single origin. The procentriole does not initiate a procentriole, because it is unmodified by PLK1, and thus is not competent to recruit the origin proteins.
  
  
• (iii) At the G2/M transition, PLK1 modification of procentrioles makes them competent for recruiting pericentriolar material proteins involved in microtubule nucleation and organization, such as γ-tubulin [57,94]. Once this happens, centrioles become competent to organize microtubule-organizing centres and duplicate in the next cell cycle.
  
  
• (iv) At mitosis, the two pairs of centrioles, each consisting of a mother centriole and an engaged procentriole, associate with the mitotic spindle. Passage through mitosis licenses the mother centriole by disengaging the procentriole, allowing a new focus to form, or the old to be re-used, and licenses the daughter by PLK1-dependent modification. Thus, in the ensuing G1, both centrioles received by a cell are competent for duplication.
  
  

Choosing sides – asymmetric centriole and basal body assembly 2

Centrioles and basal bodies (CBBs) are characterized by their nine sets of triplet microtubule ‘blades’ that are arranged in a cylindrical structure (Fig. 1A). Although the two structures are analogous in their triplet microtubule organization, centrioles and basal bodies have unique functional roles (Fig. 1B,C). Centrioles function at the centrosomes to organize cellular microtubules, whereas basal bodies organize the microtubules of cilia. Centrosomes are comprised of a pair of centrioles surrounded by the pericentriolar material (PCM), which possesses microtubule-nucleating activity (Fig. 1B). Moreover, centrioles and basal bodies interchange their functions during the cell cycle when a centriole transitions to a basal body during G0/G1 to organize the primary cilium (Fig. 1C). Once cells enter the cell cycle, the cilium is resorbed and the basal body returns to its role as a centriole at the centrosome. The term centriole, basal body or CBB refers to when these structures are functioning at centrosomes, cilia or both, respectively. During the canonical cell cycle, duplication of new centrioles initiates at the G1/S-phase boundary of the cell cycle, producing a single centriole assembly event per centriole to ensure that future daughter cells are provided with two centrioles; one old mother from the previous cell cycle and one new daughter centriole from the current cell cycle.






Overview of CBB structure and function. 
(A) Structural organization of CBBs. The cartwheel (CW) resides at the proximal end with the minus ends of the polarized triplet microtubules (red). Microtubule plus ends orient towards the distal end. A diagram of a cross section through the cartwheel is shown underneath. 
(B) Image and schematic representation of centrioles (centrin in red) and PCM (γ-tubulin in green) in retinal pigmented epithelial cells (RPE1). M, mother centriole. D, daughter centriole. Scale bar: 0.5 µm. 
(C) In the image, the basal body is labeled in red using an antibody against centriole and spindle-associated protein (CSAP) as described previously (Backer et al., 2012) and the primary cilium is stained for Arl13B in green. M, mother basal body; D, daughter centriole. Scale bar: 0.5 µm. 
(D) General schematics of the orientation of new centriole assembly. M, mother centriole; D, daughter centriole. The diagram shown contains a stalk linker (purple), which attaches the wall of the mother centriole cylinder to the daughter pro-centriole through the cartwheel. (E) New basal bodies form at a defined triplet microtubule on the mother basal body in Chlamydomonas. Tomographic reconstruction of pro-basal bodies (blue) forming specifically at triplet microtubule (in purple) number 8 (asterisks) of the mother basal. The rootlet microtubules (shown in red) define the position of each triplet blade. Scale bar: 200 nm. Image taken from O'Toole and Dutcher (O'Toole and Dutcher, 2013) with permission.




Vertebrate centriole and PCM organization and assembly. 
(A) Modular organization of the PCM into toroidal domains. The C-termini of pericentrin (PCNT; in blue) are positioned near the centriole triplet microtubule blades, whereas the N-termini project away from the centriole. Concentric rings of protein domains localize to the surrounding architecture with CEP120 (green) closest and γ-tubulin (dark red) furthest from the centriole wall. Image taken from Lawo et al. (Lawo et al., 2012) with permission. 
(B) The PCM ‘molecular gate’. The gap (shown in the image on the right) is thought to accommodate the newly forming centriole (marked with Sas-4, green). Shown here are different views of the volume rendering of Drosophila PCM organization in G2 cells with PLP in red and the centriole marker Sas-4 in green. Image adapted from Mennella et al. (Mennella et al., 2012) with permission. 
(C) Model of PCM organization around centrioles during the cell cycle highlighting centriole and PCM dynamics. In G1, the PCM retains a toroidal organization and, during S-phase, the nascent centriole is accommodated by loss of one PCNT/PLP cluster. Centriole duplication occurs through CEP152 and CEP192-dependent loading of PLK4 at a single focus, where new centriole biogenesis will initiate and begin with cartwheel formation. Upon mitotic entry, the PCM expands its general organization and γ-tubulin-dependent microtubule nucleation capacity. The illustration is based on work described in Fu and Glover, 2012, Lawo et al., 2012, Mennella et al., 2012 and Sonnen et al., 2012.







1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113104/
2) http://jcs.biologists.org/content/127/13/2803

Building a ninefold symmetrical barrel: structural dissections of centriole assembly 1

Centrioles are short microtubule-based organelles with a conserved ninefold symmetry. They are essential for both centrosome formation and cilium biogenesis in most eukaryotes. A core set of five centriolar proteins has been identified and their sequential recruitment to procentrioles has been established. However, structures at atomic resolution for most of the centriolar components were scarce, and the underlying molecular mechanisms for centriole assembly had been a mystery—until recently. In this review, I briefly summarize recent advancements in high-resolution structural characterization of the core centriolar components and discuss perspectives in the field.

 Introduction

Centrioles are cylindrical cellular structures present in almost all eukaryotic lineages. They are 0.1–0.5 µm long and 0.1–0.2 µm in diameter, and are usually composed of nine microtubule triplets at their outer wall [1,2]. Exceptions to this organization are found in Drosophila embryos (nine doublet microtubules), and in Caenorhabditis elegans sperm cells and early embryos (nine singlets).
Centrioles usually exist in pairs, oriented perpendicularly to one another, and are surrounded by the pericentriolar material (PCM) to form the centrosome [3,4]. The PCM has a layered structure made of fibres and matrix proteins, and is responsible for microtubule nucleation and anchoring [5–8]. As such, the centrosome is the major microtubule organizing centre of animal cells. Centrosome duplication is controlled by centriole replication, which in most animal cells is tightly coordinated with the mitotic cycle. Miscoordination of the two processes often leads to alterations in centrosome number and/or structure that result in chromosome missegregation and severe consequences such as cancerous growth of the cell [9–12]. Besides nucleating centrosomes, centrioles also play a pivotal role in the biogenesis and operation of the cilium, an antenna-like structure projecting out from the cell surface [13]. Cilia are present on virtually every human cell type, and defects in their assembly or function lead to a plethora of human disorders, which are collectively called the ciliopathies [14,15].

Despite cell biologists' great interest in the centriole and extensive cellular and microscopic studies on this organelle for several decades, the molecular composition of the centriole was not fully determined until recently. Because of its favourable features for laboratory study and the availability of powerful genetic and genomic tools, the nematode C. elegans has become an important model organism in studying centrosome duplication and centriole assembly. Studies in several research groups have uncovered five core C. elegans centriolar proteins: the polo-like kinase ZYG-1 [16], and the four coiled-coil-containing proteins, SPD-2 [17,18], SAS-4 [19,20], SAS-5 [21] and SAS-6 [22,23]. All of these proteins contain both structured domains and disordered segments and are conserved in all ciliated cells.


Centriole duplication is a multistep process, with each of the five centriolar proteins being recruited in a hierarchical manner [24,25]. In C. elegans, centriole assembly is initiated by the recruitment of SPD-2 to the proximal side of the mother centriole. The kinase ZYG-1 is then incorporated into the SPD-2 scaffold, and is required for the subsequent recruitment of the SAS-5/SAS-6 complex. SAS-4 is then recruited to add an outer tube around the central tube formed by SAS-5 and SAS-6. Finally, nine singlet microtubules are assembled onto the outer tube to generate a daughter centriole (figure 1). In other organisms, including Drosophila and humans, additional essential proteins besides the five core components are also recruited during centriole assembly (figure 1).




Recent years have seen a surge of structural dissections of the centriole, with an increasing number of high-resolution structures reported for most of its core components. Together, these have greatly advanced our understanding of centriole duplication at the atomic level. Nevertheless, a full molecular view of centriolar assembly is a mission that is not yet completed. This review briefly summarizes recent progress in the structural characterization of centriole assembly, which promises a challenging but rewarding future.

SPD-2/Cep192 and Asterless/Cep152

Centriole duplication in animals is initiated by recruiting the centriolar receptors SPD-2 and/or Asterless to the vicinity of the mother centriole. These receptors then recruit polo-like kinase 4 (Plk4) and ZYG-1, depending on which organism is under consideration (figure 2a). SPD-2 is a coiled-coil-containing protein localizing to both the centriole and the PCM in C. elegans [17,18]. It has an N-terminal acidic region that binds to the cryptic polo-box (CPB) domain of ZYG-1 (figure 2b) [26]. The Drosophila orthologue of SPD-2 lacks the acidic region for Plk4 binding, and thus is dispensable for centriole duplication [27,28] (figure 2b). The mammalian SPD-2 orthologue Cep192 also bears an acidic region that interacts with the Plk4 CPB domain, but it is located a bit further downstream in the sequence (figure 2b).



Recruitment of the polo-like kinases Plk4/ZYG-1 and domain organization of the centriolar receptors.
(a) Different recruitment modes of ZYG-1/Plk4 by their respective centriolar receptors SPD-2/Asterless. Caenorhabditis elegans has only one centriolar receptor, SPD-2, to recruit ZYG-1. Drosophila has both SPD-2 and Asterless, but only Asterless can bind and recruit Plk4. In humans, both SPD-2 and Asterless bind Plk4 but in a mutually exclusive manner to temporally and spatially regulate Plk4 recruitment. 
(b) Domain organization of SPD-2 homologues. Residue numbers of domain boundaries are shown above the schematics. (c) Domain organization of Asterless and Cep152.

Asterless was originally identified in Drosophila as a protein essential for aster formation during male meiosis [29]. The protein was later revealed to be a constitutive centriolar component involved in the initiation of centriole duplication [30–33]. Cep152, the mammalian orthologue of Asterless, is also involved in both centriole duplication and PCM recruitment [34–36]. Both Drosophila Asterless and human Cep152 bind to the CPB domain of Plk4 via their N-terminal acidic region [32,35]. Recent studies demonstrated that Plk4 additionally binds to the C-terminal region of Asterless, which serves to stabilize Plk4 during mitosis [37].
Although it is now well established how SPD-2 and Asterless interact with their kinase partners ZYG-1 and Plk4, they have been structurally less characterized. Bioinformatics analysis suggests that all SPD-2/Cep192 orthologues contain a mostly unstructured N-terminal region with a short coiled-coil domain and a long C-terminal β-strand-rich region (figure 2b). By contrast, all Asterless/Cep152 orthologues contain mostly helical structures that are predicted to form coiled-coils, with no β-strand content (figure 2c). Caenorhabditis elegans and mammalian SPD-2, and Drosophila and mammalian Asterless, also contain a highly acidic region towards their N-termini, which interacts directly and tightly with their kinase partners [26]. This tight interaction creates something of a puzzle however, as the receptors and the kinases do not seem to form a robust complex in the cell until the receptors are targeted to mother centrioles. Recent studies by multiple imaging/biophysical techniques have shown that C. elegans SPD-2 is monomeric in the cytoplasm [38]. It was postulated that the acidic region for kinase binding might be concealed via its interaction with another (probably basic) part of the same protein; receptors targeted to the nascent centriole might then release such auto-inhibition to facilitate interaction with the downstream kinases [26].

Another unresolved issue about the centriolar receptors (SPD-2 and Asterless) in centriole assembly is how they themselves are recruited to the mother centriole. One possibility is that Plk1, another critical polo-like kinase linking centriole biogenesis to cell-cycle progression [39–41], might phosphorylate SPD-2/Asterless to promote their targeting and the initiation of centriole assembly. In human cells, co-localization of another coiled-coil-rich protein Cep63 with Cep152 in a ring-like structure around the proximal end of the mother centriole has been shown [42]. Cep63 knock-out leads to both Cep152 mislocalization and deficiency in procentriole assembly. It was hypothesized that Cep63 might oppose the activity of Plk1 that modifies centrosomal Cep152 [42]. From the structural point of view, it would be interesting to determine the high-resolution structures of the C-terminal β-strand-rich region in SPD-2 and investigate how they are involved in centriole duplication (figure 2b). To determine the high-resolution structures of Asterless and Cep152 might prove challenging because of their high coiled-coil content (figure 2c). Reconstitution of a complex of the coiled-coil with an interaction partner might help to make its structural studies amenable. The MitoCheck consortium has reported that at least nine other proteins interact with Cep152 in mammalian cells [43].

Plk4/ZYG-1

In metazoans, the serine/threonine kinase Plk4 controls daughter centriole assembly and couples centriole duplication with cell-cycle progression [44,45]. As the master regulators of centriole assembly, Plk4 and ZYG-1 are essential for the recruitment of downstream proteins to the procentriole [24,25,46]. In C. elegans, ZYG-1 directly binds SAS-6 in order to recruit it and its interaction partner SAS-5 to the mother centriole [47]. In Drosophila and mammals, Plk4 phosphorylates Ana2/STIL to trigger the direct interaction between Ana2/STIL and SAS-6, which is essential for their recruitment during centriole assembly [48–51]. Plk4 is the most divergent member of the polo-like kinase family, and C. elegans ZYG-1 is one of the most divergent of Plk4 orthologues [52]. Despite the high divergence in their primary sequences, ZYG-1 and Plk4 share a similar structural arrangement consisting of three structural domains: the N-terminal kinase domain, the central CPB domain and the C-terminal single polo-box (PB3) domain (figure 3a).





Structural dissection of Plk4/ZYG-1. 
(a) Schematic showing domain organization of Plk4/ZYG-1.
(b) Ribbon diagram of the crystal structure of the human Plk4 kinase domain loaded with an ATP analogue (PDB code: 3COK). The structure is colour-ramped from blue (N-terminus) to red (C-terminus). The bound nucleotide is shown as sticks. 
(c) (i) Ribbon diagram and (ii) corresponding electrostatic surface plot of the crystal structure of the Drosophila Plk4 CPB dimer (PDB code: 4NK7). 
(d) (i) Ribbon diagram and (ii) corresponding electrostatic surface plot of the crystal structure of the ZYG-1 CPB dimer (PDB code: 4NKB). 
(e) Crystal structure of human Plk4 CPB in complex with the acidic region of SPD-2 (dark blue) (PDB code: 4N7Z). (f) Crystal structure of human Plk4 CPB in complex with the acidic region of Asterless (pink) (PDB code: 4N7 V). (g) Ribbon diagram of the crystal structure of the mouse Plk4 PB3 (PDB code: 1MBY). Different molecules in all ribbon diagrams are coloured differently. Positive and negative charges in the electrostatic plots are shown in blue and red, respectively.

The kinase domain of Plk4 adopts a similar conformation to that of Plk1, with the nucleotide binding site located in the cleft between two lobe-like modules (figure 3b). The central and C-terminal regions of Plk4 and ZYG-1 are the least similar in terms of protein sequence comparison. It has been debated whether ZYG-1 is a bona fide Plk4 or a structurally distinct functional orthologue [44]. To address this, it was critical to compare their respective CPB and PB3 domains to find out how similar they really are in three-dimensional structure. Initially, a crystal structure of the Drosophila Plk4 CPB was determined, based on which a side-by-side dimeric conformation was proposed [53]. However, this orientation of the Plk4 CPBs was later found to be a crystallographic artefact [26,54]. Other more recent structural studies on the ZYG-1 and Plk4 CPBs revealed that they in fact form a similar Z-shaped dimer, with a basic patch across the dimeric interface for binding by SPD-2 and/or Asterless [26,55]. Interestingly, despite the high similarity in their overall conformation, there are subtle structural variations in the CPBs of ZYG-1 and Plk4 that confer their selective binding to the respective receptors [26]. The CPB of Drosophila Plk4 bears a flat and broad basic patch (figure 3c), whereas the basic patch on the ZYG-1 CPB is narrow and elongated because of the expanded angle between the arms (figure 3d). A crystal structure of the human Plk4 CPB revealed that it is very similar to that of the Drosophila Plk4 CPB [55]. The structure of the human Plk4 CPB in complex with either SPD-2 (figure 3e) or Asterless (figure 3f) showed that the two receptors are bound with distinct modes, suggesting an intricate spatio-temporal regulation of Plk4 recruitment [55].

A crystal structure of the C-terminal PB3 of mouse Plk4 showed that it forms a dimeric structure distinct from the canonical polo-box (figure 3g) [56]. Recent studies suggested that the PB3 of human Plk4 plays an important regulatory role by relieving auto-inhibition of the kinase domain [57]. Similarly, the C-terminal PB3 in ZYG-1 has also been shown to play a regulatory role in its function. ZYG-1 deletion mutations lacking its PB3 make the kinase behave differently in mitosis and meiosis [58]. It was proposed that the ZYG-1 C-terminus may possess two distinct regulatory roles: one for inhibiting the kinase activity and the other for differential regulation of ZYG-1 recruitment in mitosis and meiosis [58]. However, the underlying mechanism for how the PB3 regulates ZYG-1 activity remains elusive. A crystal structure of the mouse Plk4 PB3 dimer revealed a deep interfacial pocket between the two molecules, which was proposed to be a potential ligand-binding site [56]. However, no ligands binding to this pocket of the Plk4 PB3 have yet been identified. Recent studies further demonstrated that the human Plk4 PB3 does not dimerize [57]. Therefore, it remains unclear whether the PB3 of Plk4 in different organisms indeed all forms a dimer and, if so, whether the ZYG-1 PB3 shares a similar conformation to it. Determination of high-resolution structures for the PB3 domains of ZYG-1 and various Plk4s would also help investigate how PB3 mechanistically regulates the function of Plk4 and ZYG-1 in vivo.

SAS-5/Ana2/STIL and SAS-6

SAS-5 and SAS-6 are co-dependent for their centriolar localization, which is essential for the subsequent recruitment of SAS-4 [23]. SAS-5 is functionally orthologous to Drosophila Ana2 and vertebrate STIL, with a 90-residue STIL/Ana2 (STAN) motif at the C-terminus [59]. Reflecting evolutionary history, this motif is more conserved among vertebrates and flies (greater than 30% identity) than with the corresponding region in C. elegans SAS-5 (12% identity). Likewise, the binding modes of Ana2 and SAS-5 to their respective partners seem to be different. While the binding of SAS-5 to SAS-6 is mediated via synergistic hydrophobic and electrostatic interactions between the C-terminal 15 residues of SAS-5 (which is predicted to form a short α-helix) and a central region of the SAS-6 coiled-coil [60], such an interaction seems to be absent in Drosophilaand mammals. This is despite the fact that both Ana2 and STIL are also predicted to possess a short helix towards their C-termini and that two of the four residues at the C-terminus of SAS-5 for SAS-6 binding are also conserved in this helix of Ana2 and STIL. Instead, the interaction between Ana2/STIL and SAS-6 was recently revealed to be mediated by the Plk4-dependent phosphorylation of a number of conserved serine/threonine residues in the STAN motif of Ana2/STIL [48–51]. ZYG-1 was initially reported to phosphorylate SAS-6 at serine 123 residue [61]. However, later studies with mutations of 38 potential phosphorylation sites in C. elegansSAS-6 (including serine123) suggest that SAS-6 is unlikely to be the phosphorylation target of ZYG-1 [47]. It remains to be determined whether ZYG-1 phosphorylates SAS-5 in a similar manner to that of Plk4 on Ana2/STIL.

Studies using various biophysical methods suggested that the central region of SAS-5 forms a tetramer, which may function as a cross-linker to strengthen the higher-order structure formed by SAS-6 [62]. Detailed molecular mechanisms underlying SAS-5 oligomerization have been revealed very recently. Crystal structures show that SAS-5 contains two independent oligomeric domains: a coiled-coil region spanning residues 125–180 and an Implico domain consisting of residues 210–265 (figure 4a) [63]. While the Implico motif forms a stable dimer via the intertwining of the zigzagged triple-helix structures, the coiled-coil can exist as either a dimer or trimer depending on its concentration. Together, these properties allow SAS-5 to form a higher-order assembly that may create a seeding point for SAS-6 oligomerization [63]. In the meantime, a crystal structure of the central coiled-coil domain of Ana2 has been reported, which reveals that it forms a parallel, symmetrical 4-helix bundle (figure 4b) [64]. This tetrameric form, together with its N-terminal SAS-4 binding site and C-terminal phosporylation-dependent SAS-6-binding STAN motif, was believed to actively contribute to centriole assembly by interconnecting neighbouring layers of cartwheels formed by SAS-6 [64]. While auto-assembly of both SAS-5 and Ana2 is essential for centriole biogenesis, their different oligomerization modes might reflect the morphological variations of the respective SAS-6 oligomers that have been observed (see below).




Domain arrangements and crystal structures of SAS-5 and Ana2. 
(a) Domain organization and crystal structures of SAS-5. The central coiled-coil may form a dimer or trimer depending on the protein concentration. The Implico domain forms an intertwined homodimer. The C-terminal 15 residues of SAS-5 (shown in red) are predicted to form an α-helix that binds directly to a central region of the SAS-6 coiled-coil. 
(b) Domain organization of Drosophila Ana2 and crystal structure of its parallel, symmetrical 4-helix bundle. Plk4 phosphorylates four conserved serine residues in the STAN motif to promote the strong interaction between Ana2 and SAS-6.

SAS-6 is one of the most thoroughly characterized centriolar proteins at a structural level. It is a modular protein with an N-terminal head domain followed by a long coiled-coil and an intrinsically disordered C-terminal tail (figure 5a). Numerous crystal structures of the head domain of SAS-6 from various organisms have been reported, which show a conserved intermolecular interaction between the head domains [65–68]. Furthermore, a crystal structure of the C. elegans SAS-6 coiled-coil region shows that two SAS-6 molecules form a parallel dimer via a strong interaction between their coiled-coils [60]. Together, these studies suggest that 18 SAS-6 molecules can form nine parallel homodimers that then assemble into a cartwheel-like structure. The cartwheel is stabilized through the interactions between the neighbouring head domains in the closed ring (figure 5b–d). This working model finally provides a molecular explanation for the ninefold cartwheel structure originally identified by electron microscopy [69].







Structure and assembly of SAS-6. 
(a) Domain organization and ribbon diagram representation of SAS-6. SAS-6 molecules form a parallel dimer via their coiled-coil domains. The SAS-5 binding site is indicated. 
(b) Assembly of nine SAS-6 dimers into a cartwheel-like structure via intermolecular interactions of the head domains around the ring. 
(c) Coloured schematic showing the head–head interaction between two neighbouring SAS-6 dimers. 
(d) Ribbon diagram of the crystal structure of the C. elegans SAS-6 head domain dimer (PDB code: 3PYI). Arrows point to the loops mediating the intermolecular interaction. 
(e) Three working models for formation of the cylinder-like structure by SAS-6. The spokes formed by SAS-6 coiled-coils are omitted for clarity.

While the cartwheel formed by SAS-6 is believed to be important for dictating the ninefold symmetry of centrioles, the interaction between the SAS-6 head domains is relatively weak, with a Kd of only 60–100 µM. This argues against the autonomous assembly of the cartwheel by SAS-6 alone. In the cell, this issue might be solved via the cross-linking action of SAS-5/Ana2/STIL as described above. Interestingly, it was shown recently that in human cells cartwheel assembly occurs in the proximal lumen of the mother centriole [70]. This assembly process is initiated by the recruitment of SAS-6 dimers to disengaged centrioles, and mediated by the interaction between the SAS-6 C-terminal tail and SAS-4 located at the luminal wall [70]. However, this seems to be species-specific and might be employed only by cycling cells with strictly controlled centriole number, as de novo assembly of centrioles has been observed in many other species [71–73].
To form a cylindrical centriole, the SAS-6-mediated cartwheel structure apparently has to be assembled longitudinally in the lumen of the centriole. One way to achieve this is by stacking multiple rings on top of one another, as observed in the flagellate Trichonympha (figure 5e, left) [74]. This assembly mechanism might also occur in other organisms [75,76]. The other means of SAS-6 assembly was found in C. elegans where SAS-6 forms a spiral-like structure, and two such spirals wind around each other to form the central tube of the centriole (figure 5e, middle) [65]. It is postulated that SAS-6 might also assemble in a third way, in which SAS-6 forms a single tight spiral formed by the slightly tilted hinge region between the head group and the coiled-coil (figure 5e, right) [77]. However, the single spiral model has not been seen in any real centrioles so far and its existence remains undetermined.

5. SAS-4/CPAP

SAS-4/CPAP is the last core centriolar protein to be recruited. It is located at the outer wall of the centriolar barrel and plays an essential role for subsequent recruitment of microtubules [24,25]. SAS-4/CPAP was originally identified in mammals by yeast two-hybrid screens [78]. Homologues of SAS-4/CPAP were also later identified in Drosophila and C. elegans [19,20,79]. All SAS-4/CPAP proteins share a similar overall domain arrangement, with a mostly disordered N-terminus, a central coiled-coil and a globular C-terminal T complex protein 10 (TCP) domain (figure 6a). Crystal structures of the TCP domain of SAS-4 proteins from zebrafish andDrosophila showed that it forms a long β-meander consisting of about 20 β-strands, which interacts directly with a conserved proline-rich region in STIL/Ana2—the consensus sequence of this region is PR××P×P (figure 6b) [80,81]. Interestingly, the seemingly stable TCP structure does not contain a defined hydrophobic core or flanking globular domains on either end.




Domain organization and structural dissection of SAS-4/CPAP. 
(a) Domain organization of three representative SAS-4/CPAP homologues. All SAS-4/CPAP homologues share a similar structural arrangement with a mostly disordered N-terminus, a central coiled-coil and a β-strand-rich C-terminal TCP domain. Residue numbers of domain boundaries are shown above the schematics. 
(b) Ribbon diagram of the crystal structure of the TCP domain of Danio rerio CPAP in complex with the ‘PR××P×P’ motif from STIL. The motif is shown as sticks (PDB code: 4BXR). The complex is shown in two perpendicular orientations.

While the interaction mode between the SAS-4 TCP domain and STIL/Ana2 is conserved in Drosophila and vertebrates, the interaction between C. elegans SAS-4 and SAS-5 may have diverged significantly. For instance, although the TCP domain is required for C. elegans SAS-4 to interact with the N-terminal part of SAS-5, by itself it is not sufficient [80]. Furthermore, SAS-5 does not contain the consensus TCP-binding motif (PR××P×P) present in STIL/Ana2 [80].
The function of SAS-4/CPAP in recruiting microtubules during centriole assembly has been confirmed. However, the mechanism is still unclear. One hypothesis is that the TCP domain ofDrosophila CPAP forms monomers in solution, but upon incorporation into procentrioles the TCP domain would bind to STIL towards the interior of the centriole whereas the N-terminal part of SAS-4/CPAP interacts with Asterless/Cep152 and microtubules at the outer wall [80]. Another is that the TCP β-sheet is capable of forming large assemblies via a head-to-tail interaction. This was observed in the crystal structure and was postulated to help CPAP to form continuous connections along the long axis of the centriolar outer wall. Such connections, together with the central coiled-coil of the protein, would then link the pinhead with the microtubules to stabilize the cartwheel stacks in the centriole [81]. Structural and functional analysis of Drosophila SAS-4 TCP domain by another group further confirmed the role of SAS-4 as a mediator to tether PCM complexes to the centriole to facilitate centrosome assembly [82]. However, further studies are needed to find out the exact structural roles of SAS-4/CPAP in centriole assembly.

6. Other proteins involved in centriole assembly

Besides the five core centriolar components, a number of other proteins participating in centriole duplication have recently been identified. Bld10/Cep135 is an approximately 170 kDa protein originally found in Chlamydomonas and essential for cartwheel formation [83]. It consists mostly of coiled-coils and was later found to be a major spoke-tip component, serving to bridge the cartwheel to the triplet microtubules [84]. A homologue of Bld10/Cep135 was subsequently identified in Drosophila, and was reported to play a role in the formation of both the central tube in mature primary spermatocytes [85] and the central pair of microtubules in the flagellar axoneme [86,87]. However, no Bld10/Cep135 homologue has been identified in nematodes.
The localization of Bld10/Cep135 to procentrioles depends on both Cep120 and SPICE1 [88]. Cep120 is an approximately 100 kDa protein participating in centriole assembly by asymmetrically localizing to the daughter centriole [89]. The protein contains an N-terminal C2 domain, a central region for SAS-4/CPAP binding and a C-terminal long coiled-coil for dimerization [90]. It was reported that Cep120 interacts with SPICE1, another large coiled-coil-containing protein, and the two proteins are co-dependent for their localization to procentrioles [88]. Cep120 and SPICE1 are believed to work cooperatively with SAS-4/CPAP to regulate centriole elongation [88,90].

Comparative genomics and proteomics studies of isolated basal bodies from Chlamydomonas reinhardtii have identified 18 proteins (Poc 1–Poc 18) proposed to be centriolar constituents [91]. Poc1 is one of the best-characterized Poc proteins. It contains an N-terminal WD40 domain and a C-terminal coiled-coil and, like Bld10/Cep135, is hypothesized to stabilize microtubules, and thus maintains centriole assembly and integrity [92–95].
In animal cells, a cap structure at the distal end of centrioles is formed to suppress centriole-templated ciliogenesis. The cap complex consists of CP110 and Cep97, two large proteins consisting mostly of coiled-coils [96]. Additionally, several other proteins have been identified to be more specifically involved in assembly of an appendage structure at the distal end of mother centrioles. These proteins include Ccdc41/Cep83, Cep89/Cep123, Sclt1, Fbf1 and Cep164 [97–99]. The distal appendage serves to anchor the mother centriole onto the plasma membrane during ciliogenesis.




1) http://rsob.royalsocietypublishing.org/content/5/8/150082

Pericentriolar material structure and dynamics  1

A centrosome consists of two barrel-shaped centrioles embedded in a matrix of proteins known as the pericentriolar material (PCM). The PCM serves as a platform for protein complexes that regulate organelle trafficking, protein degradation and spindle assembly. Perhaps most important for cell division, the PCM concentrates tubulin and serves as the primary organizing centre for microtubules in metazoan somatic cells. Thus, similar to other well-described organelles, such as the nucleus and mitochondria, the cell has compartmentalized a multitude of vital biochemical reactions in the PCM. However, unlike these other organelles, the PCM is not membrane bound, but rather a dynamic collection of protein complexes and nucleic acids that constitute the organelle's interior and determine its boundary. How is the complex biochemical machinery necessary for the myriad centrosome functions concentrated and maintained in the PCM? Recent advances in proteomics and RNAi screening have unveiled most of the key PCM components and hinted at their molecular interactions ( table 1). Now we must understand how the interactions between these molecules contribute to the mesoscale organization and the assembly of the centrosome. Among outstanding questions are the intrinsic mechanisms that determine PCM shape and size, and how it functions as a biochemical reaction hub.






Structural organization of the PCM. 
(a) Negative stain electron micrograph of a centrosome isolated from Chinese hamster ovary cells. One hundred and twenty-five MTs emanate from the densely staining centre. (Adapted from [2].) 
(b) Structure of a purified Drosophila centrosome as revealed by electron tomography. A ninefold radially symmetric centriole can be seen at the centre surrounded by PCM. The inset shows a magnified view of a ring-like complex found within the PCM. These complexes measured 25–30 nm in diameter and were determined to contain γ-tubulin. (Adapted from [3].) 
(c) The γ-TuRC was later isolated from Drosophila cells and analysed by electron tomography. (Adapted from [4].) 
(d) Harsh treatment of Spisulacentrosomes with potassium iodide revealed 12–15 nm wide filaments running throughout the PCM, leading to the hypothesis that a lattice-like network forms the structural foundation of the PCM. (Adapted from [5].) 
(e) The development of subdiffraction microscopy techniques allowed high precision localization of proteins within the PCM. The first picture depicts the localization of PCNT-like protein (D-PLP) in Drosophila cells determined with stochastic optical reconstruction microscopy (STORM). Comparison of the localization of numerous proteins revealed that the interphase PCM contains ordered subdomains of defined size. However, the expansive PCM that exists during mitosis is less ordered. Localization of PCNT and γ-tubulin in human cells using three-dimensional SIM is shown. (Adapted from [6–8].)

1. Pericentriolar material structure

Decades of research have pursued atomic-level resolution of the underlying pericentriolar material (PCM) structure with little avail. This is probably owing to limitations in methodology, but also to the fact that the PCM does not behave like most ordered proteinacious assemblies. In the earliest electron micrographs depicting centrosomes in situ, the PCM appeared as a densely stained amorphous mass that surrounded the highly structured centrioles [1]. Electron microscopy (EM) micrographs of centrosomes isolated from mammalian cells did little to resolve the amorphous mass any further, although these experiments definitively showed that microtubules (MTs) originate from the PCM (figure 1) [2] and that PCM integrity is sensitive to chelating divalent cations [9].

The resolution required to distinguish subdomains within the PCM would not be achieved until the implementation of EM tomography. Using this approach, in combination with immunolabelling, Moritz et al. [3] could discern gamma-tubulin-containing ring structures 25–30 nm in diameter within PCM from isolated Drosophila melanogaster centrosomes [3]. Higher resolution structures of immunoprecipitated Drosophila γ-tubulin ring complexes (γ-TuRCs) confirmed the 25–30 nm diameter of these rings and showed that they cap the minus ends of PCM-derived MTs [4]. Similar ring structures were observed in centrosomes isolated from surf clam oocytes and, intriguingly, were stripped away after exposure to potassium iodide, leaving behind an underlying skeletonized lattice of 12–15 nm wide filaments [5]. Unlike the untreated centrosomes, the salt-stripped centrosomes could not nucleate MTs. Interestingly, the ring structures reappeared and MT nucleation could be restored if the salt-stripped centrosomes were incubated in oocyte extract. Taken together, the findings from these studies hinted that the PCM comprises a porous structural scaffold onto which γ-tubulin and other soluble components from the cytoplasm are loaded.

Concurrently, scientists sought to determine the identities and biochemical properties of the proteins that construct the PCM scaffold, or the so-called ‘centromatrix’. Researchers took advantage of the curious fact that auto-immune sera taken from scleroderma patients reacted widely with centrosomes and, thus, could be used as a robust label for specific centrosome proteins in western blot and immunofluorescence assays [10]. Use of these sera revealed that the PCM is a dynamic structure and led to the identification and biochemical characterization of PCM proteins [11]. In this manner, one of the first PCM components, pericentrin (PCNT), was identified, cloned and its necessity for spindle organization described [12]. The discovery of additional core PCM components such as Cep192/SPD-2, CDK5RAP2/Cnn, Cep152/Asterless and SPD-5 in various organisms revealed that the only major similarity among PCM organizing proteins was an abundance of coiled-coil domains [13–18]. The coiled-coil motif consists of intertwined α-helices and is known to mediate protein–protein interactions [19]. Thus, it was proposed that these numerous coiled-coil domains could mediate robust inter-molecular interactions to allow formation of the centromatrix [16,20]. Whether these coiled-coil scaffold proteins per se are sufficient to assemble the centromatrix, and whether their coiled-coil domains are critical for this assembly process, has yet to be determined.

Analysis of purified centrosomes by mass spectrometry and large-scale RNAi and localization screens in Caenorhabditis elegans, Drosophila and human cells unveiled a diverse bounty of centrosome proteins [17,21–25] (table 1). Owing to the diversity and tight clustering of PCM proteins at centrosomes, it is of little surprise that electron microscopy so far could not discern structural information about the PCM. Labelling and observing individual components within the PCM promised to circumvent this problem, but the resolution limitations of conventional light microscopy and immunogold-EM only allowed the localization of the components without generating any meaningful structural insights. However, recent advances in light microscopy technology opened new possibilities for mapping PCM architecture. Four independent studies used subdiffraction light microscopy techniques, such as three-dimensional structured illumination (three-dimensional SIM) and stochastic optical reconstruction microscopy (STORM) to identify the substructures within the PCM [6–8,65]. The authors developed antibodies to label distinct epitopes of different PCM proteins and measured their distances from the outer centriole wall. A key finding was that interphase PCM proteins are distributed in concentric toroids of discrete diameter around centrioles. A subset of proteins, human CDK5RAP2, PCNT, CEP152 and the Drosophila orthologues PCNT-like protein (D-PLP) and Asterless, were shown to exhibit highly extended conformations spanning approximately 100 nm from the centriole wall to the outer toroidal domains of the interphase PCM. In stark contrast, analysis of the same proteins during metaphase revealed no ordered structures or discrete distributions. Interestingly, the co-localization between labelled PCM protein pairs was minimal, indicating that, despite the lack of apparent organization, PCM proteins still occupy distinct domains in the metaphase centrosome [7]. These findings argue that the PCM is first assembled in interphase as an ordered, compact layer immediately surrounding the centrioles that then serves as a foundation for the expansive formation of PCM towards metaphase.

Table 1.
Important proteins for PCM assembly and function. (Key PCM proteins and their homologues from humans, flies and nematodes are grouped based on their general role in PCM biogenesis and function. Scaffold proteins are believed to be involved in forming the foundation of the PCM. The effector proteins are more peripheral factors involved in MT organization. SMC_prok_B: chromosome segregation protein SMC, common bacterial type. PACT_coil_coil: PCNT-AKAP-450 domain of centrosomal targeting protein.)





Organization of the PCM at human centrosomes 2



(a) During interphase, PCM proteins are organized in concentric toroids around mother centrioles. Proteins involved in microtubule nucleation (including the γ-tubulin ring complex (γTuRC) and CDK5RAP2) are found in the outer layers, whereas PCM components such as CEP192 and CEP120 are found closer to the wall of the mother centriole, which is decorated by CPAP. Some PCM proteins such as pericentrin have an extended conformation and are organized radially, with one end close to the centriole wall and the other end extending outwards. 
(b) Upon entry into mitosis, centrosomes acquire additional PCM, which forms an extended outer matrix around the toroidal PCM. The extended mitotic matrix contains subdomains formed by pericentrin, CEP192, CDK5RAP2 and γTuRC. The spatial relationships between these proteins in the mitotic matrix are similar to their arrangement in the interphase toroids. The main features of PCM organization are conserved between humans and flies. Drosophila protein names are shown in brackets in cases were they have been mapped.

1) http://rstb.royalsocietypublishing.org/content/369/1650/20130459
2) http://www.nature.com/ncb/journal/v14/n11/fig_tab/ncb2617_F2.html

Centrosomal/Centriolar Proteins 1











A model for the role of Sas-4 in PCM formation. 
Centrosome biogenesis begins with the formation of a nascent procentriole and the assembly of centriole microtubules. S-CAP complexes form in the cytoplasm and are then tethered to a procentriole via Sas-4. The centriole elongates and matures to a functional centrosome. Sas-4 (blue); microtubules (green); PCM (grey); appendages (orange).

1) http://www.sdbonline.org/sites/fly/aignfam/centriole.htm[/size]

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles 1

Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles.

How cells organize their interior is still an open question . For instance, the size, the count, and the position of cellular substructures must be controlled to ensure proper function. Indeed, the size of many cell organelles, such as the mitotic spindle, centrosomes, and the nucleus, is correlated with cell size, bringing up the question of how cells both determine and adjust the size of their substructures . An interesting situation arises in the case of non–membrane-bound organelles, e.g., the mitotic spindle, Cajal bodies, or germ granules, where the flux of material across the interface between the organelle and the cytosol is not controlled by an enclosing membrane. Instead, these structures often consist of many different components exchanging quickly with the surrounding cytosol, while maintaining a well-defined spatial organization. This turnover suggests that elastic stresses can relax and are thus unimportant for dynamics on long time scales, which is a characteristic property of complex fluids. In the case of germ granules it has been shown that their behavior can be explained by considering them as liquid droplets . Other cell organelles are also good candidates for a description as drop-like objects. In the case of metaphase spindles, a theoretical description based on liquid crystal properties led to predictions of spindle size as a function of kinetic parameters that was confirmed experimentally. Centrosomes are examples of organelles without a membrane that can occur in varying sizes. However, the mechanisms regulating their size and their growth kinetics are not understood. Centrosomes play a key role in organizing the microtubule network of the cell, most notably the mitotic spindle during cell division . In particular, it has been shown that in the nematode Caenorhabditis elegans, centrosome size directly sets the length of the mitotic spindle . Generally, centrosomes consist of a pair of centrioles embedded in a matrix of pericentriolar material (PCM). This structure has a dynamic life cycle in cells:

Centrioles are duplicated and two centrosomes grow by accumulating PCM to organize the mitotic spindle. After anaphase, centrosomes disassemble and each daughter cell inherits one pair of centrioles after cell division. The proteins required for this centrosome cycle are known  and at least one of them was shown to be limiting for centrosome size in C. elegans . Furthermore, the centrioles also influence the assembly of PCM , many centrosome proteins turn over quickly, and the PCM is permeable and permits the diffusion of a number of proteins. The nucleation and growth of centrosomes has been thoroughly examined in the early divisions in C. elegans embryos . Here, as in all systems, centrosome growth always begins at centriole pairs. Because there are only two centriole pairs, there are only two centrosomes in a cell. After nucleation, centrosome size follows a sigmoidal growth curve . Importantly, the growth rate and the final centrosome size depend on the size of the cell. In the divisions of early C. elegans embryos, all material has been provided by the mother, and no new material is made during the course of cell divisions. Therefore, the same components are reused in subsequent divisions by a process of growth and disassembly. This has led to the hypothesis that centrosome size is determined by a limiting component that is depleted from the cytosol as centrosomes grow. Centrosomes then disassemble at the end of cell division and the components are available for the next cell cycle. Centrosome formation has all the hallmarks of a nucleation and growth process, but currently the physical nature of the centrosome and its dynamics are not understood. Centrosomes are complex objects that consist of many components. However, only a few components are required for their formation, which allows us to seek for a simplified, minimal description to highlight essential properties. In general, any theory of centrosome growth must explain the following key features:

(i) Nucleation at centrioles must be extremely reliable. Thus, there must be a mechanism to suppress nucleation in the cytosol, whereas nucleation at the centrioles must be guaranteed. 
(ii) The sigmoidal growth curve must be accounted for. 
(iii) The coexisting centrosomes must be stable, spherical, and of similar size, whereas components can both exchange with the cytosol and internally rearrange. 
(iv) The size of the centrosome must depend on cell size. In this paper, we develop a physical description that can quantitatively account for these centrosome properties

Physical Description of Centrosomes as Active Droplets 
Centrosome growth is an aggregation process of a condensed phase of PCM components, which segregate from the cytosol. The aggregation process leads to a centrosome phase that coexists with the cytosol and does rearrange internally. This implies that the centrosome phase is viscoelastic, such that on long timescales it behaves as a liquid-like material. Thus, in our picture, centrosome material phase separates from the cytosol. Because centrosome growth requires kinase activity , the most likely scenario is that two phosphorylation states of PCM components have different assembly properties (Fig. 1). The phosphorylation reaction permits the cell to regulate assembly and disassembly of centrosomes. We propose a simplified model of centrosome assembly based on the idea that PCM is made of subunits that can exist in two different forms: 

(i) building blocks that dissolve in the cytosol (we call this form A) and 
(ii) droplet material that phase separates from the cytosol and produces centrosomes (we call this form B). 

In the case of C. elegans centrosomes, these two forms could be related to different conformations of the same structural protein, e.g., spindle defect protein-5 (SPD-5). Phosphorylation mediated by kinases like PLK-1 could then change the conformation of the structural protein and thereby influence its physical properties (Fig. 1). An important question is to understand why centrosome material aggregates only around centrioles. This implies that the centrioles act as a nucleator and at the same time spontaneous nucleation in the cytosol is suppressed. Either the centrioles could act as a passive nucleator in a so-called heterogeneous nucleation while the chemical transition from form A to form B happens away from the centrioles or the centrioles themselves could act as an active nucleator by catalyzing the chemical transition from A to B. To address such questions, we next develop the basic physical equations for centrosome assembly involving diffusion, chemical transitions, and phase separation.




Discussion
It is long been known that centrosomes have an amorphous structure formed by assembly and disassembly of PCM components. However, the principles governing centrosome dynamics and the mechanisms of centrosome assembly remain unclear. What type of material is the centrosome made of? How do the subunits from the cytosol become incorporated in the PCM? Here, we show that a model based on the idea of centrosomes forming around centrioles by autocatalytic growth of a PCM droplet phase in the cytosol can quantitatively account for key features of centrosome growth in C. elegans. The model has three key ingredients: (i) The PCM components can exist either in a soluble or in an insoluble form; (ii) the interconversion between the two forms is driven by the catalytically active centrioles and by an autocatalytic chemical reaction; and (iii) the insoluble form tends to phase separate from the cytosol. This PCM phase corresponds to the droplet phase in the model. In C. elegans, the known components required for centrosome growth are the polo-like kinase PLK-1 (14) and the coiled-coil proteins SPD-5 and SPD-2 (21, 22). It seems likely that SPD-5 and SPD-2 are proteins that can phase separate from the cytosol to form the PCM matrix after a phosphorylation reaction and that the kinase PLK-1 mediates this conversion. More experiments are needed to assess the biochemical details and identify the nature of the autocatalytic mechanism. To test



1) http://www.mpipks-dresden.mpg.de/mpi-doc/julichergruppe/julicher/CaADoPMObC2014.pdf

Procentriole assembly revealed by cryo-electron tomography 1

we observed that each one of the nine microtubule triplets grows independently around a periodic central structure. The proximal end of the A-microtubule is capped by a conical structure and the B- and C-microtubules elongate bidirectionally from its wall.






 These observations suggest that the gamma tubulin ring complex (γ-TuRC) has a fundamental role in procentriole formation by nucleating the A-microtubule that acts as a template for B-microtubule elongation that, in turn, supports C-microtubule growth. This study provides new insights into the initial structural events involved in procentriole assembly and establishes the basis for determining the molecular mechanisms of centriole duplication on the nanometric scale.

Numerous electron microscopy studies have established that centriole duplication begins at the G1/S transition when one procentriole appears next to the proximal end of each mature centrioles and elongates during late S/G2 phase, reaching their full length during the next cell cycle (Kuriyama and Borisy, 1981;Vorobjev and Chentsov Yu, 1982; Chrétien et al, 1997). Despite the recent discovery of proteins that have essential roles in centriole duplication (Strnad and Gonczy, 2008; Bettencourt-Dias and Glover, 2009), structural data are available only for Caenorhabditis elegans in which some details of the early procentriole formation steps have been revealed (Pelletier et al, 2006). First, a 60-nm-long central tube, oriented perpendicular to the wall of the mother centriole, is formed. Second, the diameter and the length of this tube increase while microtubules assemble around its circumference. The first step depends on SPD-2, ZYG-1, SAS-5, and SAS-6, whereas the second step involves SAS-4. However, C. elegans centrioles are singlet microtubules organised around a tube and differ from the triplets in mammalian centrioles, which are supposed to organise around a cartwheel (Cavalier-Smith, 1974; Azimzadeh and Bornens, 2007).

In this study, we report the structural morphogenesis of human procentriole. We show that procentriole organise around a cartwheel, composed by a periodical central hub. Our results describe the microtubule triplet formation. The A-microtubule is nucleated by a gamma tubulin ring complex (γ-TuRC)-like structure, whereas the B- and C-microtubules are formed from the wall of A- and B-microtubules, respectively, and grow bidirectionally. In addition, each of the nine microtubule triplets grows independently around this periodic central structure.

To classify our tomograms, we took advantage of basal-body assembly studies, which revealed that centriolar wall formation starts with singlet, then doublet, and finally triplet of microtubules (Dippell, 1968). 

The central hub of the cartwheel is a periodic structure

A 100-nm central structure having a 20-nm diameter at the proximal end of the procentriole is observed from the earliest stage of duplication (procentriole with one microtubule) until late duplication stage (procentriole with nine long microtubule triplets; Figure 2A and B; Supplementary Table I). This central structure is reminiscent of the central hub of the basal body cartwheel (Dippell, 1968; Cavalier-Smith, 1974), but the radial spokes cannot be distinguished in our data, probably due to the resolution of 4 nm. Moreover, a 110-nm stalk seemed to connect the central hub to the parent centriole (Figure 2A and B;Supplementary movie 2). However, the cartwheel and the stalk are visible only in few tomograms probably due to the high protein density of PCM.



Figure 2
Visualisation of the initial procentriole structures.
(A) Z-section of a tomogram showing the central structure of the cartwheel and the connecting stalk (boxed in yellow). 
(B) Magnified view of the boxed region in (A), showing the central tube of the cartwheel (purple arrowhead) and the connecting stalk (red arrowhead). 
(C,E) Projection images of 23 Z-sections from two cryo-tomograms containing the central cartwheel structure. 
(D, F) Profile plots obtained from corresponding images (dotted lines) in (C, E). Maxima appear each 11.75 nm (with a s.d. of 2.5 and 1.16 nm, respectively). Scale bars: 100 nm (A) and 20 nm 
(B, C, E).

A γ-TuRC-like stucture is required for the nucleation of the A-microtubule

In the nascent procentriole, singlets corresponding to the A-microtubule were observed (Figures 3A and Band 4A and B). Interestingly, all A-microtubules appeared to be closed at their proximal end, whatever procentrioles showed singlet, doublet or triplet microtubules. (Figure 3A and E; Supplementary Table I). However, in fully developed mature centrioles this cap was no longer present (Figure 3G). This cap displays a conical shape and presents an asymmetry reminiscent of the γ-TuRC structure (Moritz et al, 2000; Zhang et al, 2000). This structure is strikingly similar to that found adjacent to the spindle pole body in budding yeast (O'Toole et al, 1999), and to the minus end of microtubules nucleated from Drosophila melagonaster centrosomes or from isolated γ-TuRC (Moritz et al, 1995, 2000). Therefore, it seems likely that this cap-like structure, observed here at the proximal end of the A-microtubule, corresponds to the γ-TuRC. To the best of our knowledge, this is the first time that a γ-TuRC-like structure has been described at the proximal end of the centriolar microtubule, suggesting its crucial involvement in nucleating and stabilising the A-microtubule. In fact, γ-tubulin and nedd1 recruitment of γ-TuRC have been shown to be essential for centrosome duplication (Ruiz et al, 1999; Garreau de Loubresse et al, 2001; Shang et al, 2002;Dammermann et al, 2004, 2008; Haren et al, 2006). Moreover, immuno EM labelling of γ-tubulin has been performed previously on isolated centrosomes after post-embedding (Moudjou et al, 1996) or on freeze-substituted samples (Fuller et al, 1995). Both studies reported gold particles close to the proximal end of the mature centriole, suggesting that the procentriole formation was requiring γ-tubulin.






Figure 3
Procentriole microtubule triplet growth. 
(A) Microtubule singlet of a procentriole. The proximal part of the microtubule is capped by a conical structure. The right panel is a 3 × magnification of the boxed region. 
(B) Two Z sections of an A-microtubule (singlet) spaced by 5 nm. The distal part of the microtubule forms an outwardly curved extension (open arrow). 
(C–E) Microtubule doublets from three different tomograms. Under each panel, a schematic representation of microtubule organisation is shown: A-microtubule is in purple, B-microtubule is in red (C). The B-microtubule is 35 nm from the tip of the A-microtubule cap. (D) The B-microtubule starts at 18 nm from the tip of the A-microtubule cap and shows an outwardly curved extension at its proximal extremity (open arrow). (E) The B-microtubule is at the level of the tip of the A-microtubule cap. 
(F) Microtubule triplet of a procentriole. The C-microtubule is attached to the side of the B-microtubule at a distance of 46 nm from the distal tip of the cap. The distal and proximal extremities of the C-microtubule display curved extensions (open arrows). 
(G) Microtubule triplet of a mature centriole. The microtubules are opened at their distal and proximal extremities. (F, G) Next to the panel, a schematic representation of microtubule organisation is shown: A-microtubule is in purple, B-microtubule is in red, and C-microtubule is in green. Scale bar: 20 nm.



Figure 4
The nine microtubule blades of the centriolar barrel. 
(A) Microtubule singlets present in a procentriole. Note that the length of each microtubule differs and is not arranged in order of size around the centriolar wall. Next to the panel, a schematic representation of the microtubule organisation in the centriolar wall is shown. A-microtubules are represented by a purple circle and numbered. 
(B) Procentriole displaying singlet or doublet microtubules. Note that three microtubule blades are not yet formed (2, 3, 7), whereas the blade 4 already shows a doublet microtubule. Next to the panel, a schematic representation of the microtubule organisation in the centriolar wall is shown. Present A-microtubules are represented by a purple circle and numbered, absent A-microtubules are in light purple, and B-microtubule is in red. Scale bar: 20 nm. 
(C, D) The length of the A-microtubule is measured from the proximal tip to its distal end. (C) Procentriole with nine blades of singlet (blade 5 and 6) or doublet microtubules. Note that the length of the A-microtubule is variable (in purple). The B-microtubule (in red) already reaches a length of 180 nm in blade 9, whereas the others are only 60 nm long (blade 1 or 2) or have not yet assembled (blades 5 and 6). 
(D) Procentriole with nine blades of doublet or triplet microtubules. Note that the C-microtubule (in green) in blade 8 is absent.

In contrast to the closed proximal ends of the A-microtubules, distal ends were open and showed a frequent long and slightly outwardly curved extension (Figure 3B). Such a microtubule structure was previously described for growing microtubules in vitro (Chrétien et al, 1995) and in vivo (Koning et al, 2008). Taken together, our ex vivo results suggest that the procentriolar A-microtubule grows unidirectionally from the proximal (minus) to the distal (plus) end, and are nucleated by a γ-TuRC like structure. This observation is in contrast with that of Pelletier et al (2006) in C. elegans, in which microtubules did not seem to grow preferentially at the distal or proximal extremities, though a slight positional bias for the distal region of the central tube was observed. This difference may reflect divergent mechanisms of assembly between the human and C. elegans centrioles, which involve the formation of a cartwheel structure in the former case and a tube in the later.

The A- and B-microtubules act as templates for the bidirectional growth of the B- and C-microtubules, respectively

In contrast to the A-microtubule, which was always capped at its proximal end, B- and C-microtubules were never closed at their extremities (Figure 3C and G; Supplementary Table I).






This suggests that γ-TuRC may not be required for B- and C-microtubule nucleation. In addition, the proximal extremity of the B-microtubule was found at different heights with respect to the A-microtubule tip (Figure 3C and E), which was as far as 60 nm from the cap tip in several blades, at the same height of the tip, or even below in others (Supplementary Figure S2A and C). In addition, both the proximal and distal extremities of the B-microtubule showed outwardly curved extensions in growing procentriole (Figure 3D and F), suggesting that elongation took place at both extremities. Interestingly, the proximal end of the B-microtubule appeared blunt when it reached the proximal extremity of the A-microtubule (Figure 3E and F), whereas the distal end remained curved. This observation suggests that the B-microtubule no longer grows towards the proximal end but continues growing at the distal end. Similar observations were made for the C-microtubule (Figure 3F). These observations suggest that B- and C-microtubules are nucleated without γ-TuRC and that the A- and B-microtubules act as templates for the bidirectional growth of the B- and C-microtubules, respectively. Moreover, all microtubule triplets in the mature centriole were blunt and open at their proximal extremity (Figure 3G), suggesting that the γ-TuRC is no longer necessary and is removed from the A-microtubule ends when microtubule blades are fully developed and stabilised. This stabilisation has been attributed to tubulin modifications (Bobinnec et al, 1998) or to the presence of rare tubulins, such as δ- and ɛ-tubulins (Goodenough and StClair, 1975; Dupuis-Williams et al, 2002; Dutcher, 2003). In addition, the distal extremity of the microtubule triplet does not present outwardly curved extensions in agreement with the fact that mature centriole does not grow anymore (Kochanski and Borisy, 1990).





1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876950/

SPD2/ CEP192

The Mammalian SPD-2 Ortholog Cep192 Regulates Centrosome Biogenesis 1

Cep192 Is Required for Mitotic Spindle Assembly

In order to gain insight into the potential function of Cep192 in centrosome biogenesis, we depleted Cep192 in HeLa cells by using enzymatically prepared small interfering RNAs (esiRNAs) . Transfection of Cep192 esiRNA resulted in a marked reduction of Cep192 levels either in whole-cell extracts (Figure S5) or at mitotic centrosomes (Figures 2A and 2B) to less than 5% those of the control. A significant decrease in a high-molecular-weight band corresponding to Cep192, which migrates slower than its predicted molecular weight of 192 kDa, was observed . Cep192 depletion led to a 4-fold to 5-fold increase in the mitotic index relative to the control, consistent with our previous results (Figure 2C) . This increase in mitotic index is explained by a high incidence of aberrant spindles upon Cep192 RNA interference (RNAi) (>80%), most of which (>90%) are monopolar (Figures 2A and 2D). Similar results were obtained with chemically synthesized siRNAs, although higher mitotic indices (15%–20%) were observed (Figure S2). To rule out potential off-target effects , we generated stable cell lines by BAC transgenesis expressing an RNAi-resistant mCep192::GFP messenger RNA (mRNA). In the two clones tested, we observed no increase in mitotic indices or abnormal mitotic spindles after endogenous Cep192 depletion (data not shown). These results showed that mCep192::GFP can functionally complement endogenous Cep192, thereby validating the specificity of our Cep192 RNAi phenotype (Figure S2).

Cep192 Is Required for PCM Recruitment and Centrosome Maturation

The spindle assembly defect observed upon Cep192 RNAi suggests that it might be required for centrosome assembly and/or centrosome maturation. Consistent with this possibility, we observed that the depletion of Cep192 lead to a decrease in γ-tubulin and NEDD-1 on interphase centrosomes, whereas Pericentrin levels were not significantly affected (Figure 3A). Consistent with the known role for NEDD-1 in γ-tubulin ring complex (γ-TuRC) recruitment and microtubule nucleation from interphase centrosomes, we observed a marked defect in microtubule repolymerization after recovery from cold treatment in Cep192-RNAi-treated cells (Figure 3B) . Together, these results suggest that Cep192 depletion impairs NEDD-1 recruitment to interphase centrosomes, which in turn causes a reduction in γ-TuRC recruitment, which then causes the observed defects in microtubule nucleation .

Conclusions

We have shown that Cep192 is a pericentriolar protein that accumulates at centrosomes during mitosis and is required for PCM recruitment, centriole duplication, microtubule nucleation, and centrosome maturation. At the molecular level, how does Cep192 carry out these functions? A clue might reside in our observation that NEDD-1, a protein required for all of these processes, is dependent on Cep192 to accumulate at centrosomes. Therefore, an important function of Cep192 might be to participate in the centrosomal recruitment of NEDD-1 

1) http://www.cell.com/current-biology/fulltext/S0960-9822(08)00002-X

Asterless / CEP152

Conserved Molecular Interactions in Centriole-to-Centrosome Conversion 2

Abstract

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signaling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully-elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of 


Cep135, 
Ana1:
Cep295 and 
Asterless:


Cep152 onto daughter centrioles during mitotic progression. This generates a molecular network spanning from inner- to outer-most parts of the centriole. Ana1 forms a molecular strut within the network and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless:Cep152, partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood.

Loss of asl function impedes the stabilization/maintenance of PCM at the centrosome. 1 In embryos deficient for Asl, development is arrested right after fertilization.

1) http://www.sdbonline.org/sites/fly/genebrief/asterless.htm
2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719191/

Electric fields generated by synchronized oscillations of microtubules, centrosomes and chromosomes regulate the dynamics of mitosis and meiosis  1

https://reasonandscience.catsboard.com/t2090-centriole-biogenesis-and-the-duplication-cycle-amazing-evidence-of-design#4888

Background 
The choreography of microtubules, centrosomes and chromosomes during mitosis and meiosis is beautifully designed by nature. Finely regulated and synchronized movements of these super-macromolecular complexes against the entropic forces within a dividing cell ensure the fidelity of the genetic material in both daughter cells. Currently, several models exist for the mechanisms of chromosome congression and spindle body assembly during M phase such as the search and capture model, kinetochoremediated k-fibre formation, kinetochore motors contributing to congression, and the polar wind model. The mechanisms evoked by these models probably overlap, so there is redundancy among them, since mutations in the genes involved have only mild effects on chromosome congression during mitosis [1]. Many open questions remain within these models. In the polar-wind model, an unknown force (also known as the ejection force) generated by the spindle poles is considered to push the chromosomes to the spindle equator. Laser microsurgery experiments show that chromosome fragments without kinetochores are invariably expelled from the spindle, and chromosomes without kinetochores can still move from the vicinity of the spindle pole to the spindle equator . The ejection force of the spindle body is dependent on the polymerization of spindle body microtubules, as depolymerization of astral microtubules by nocodazole or colcemid prevents the expulsion of severed chromosome arms from the spindle, whereas stabilization of microtubules by taxol drives chromosomes to the periphery of the astral array. In addition, the driving force responsible for the pole-ward flux of spindle microtubules during metaphase remains uncharacterized. Cellular electric fields have been studied in various cell types, and several studies have reported the existence of dielectrophoretic forces around cells; electromagnetic interactions between cells have also been studied. Cifra et al. proposed that microtubules, which comprise heterodimers polymerized into a helical structure, can generate an electric field under intracellular energy excitation. Inhibition of microtubule polymerization by an external electromagnetic field has been reported by Kirson et al.. Pokorny´ et al. detected four peaks of electric field activity around yeast cells during M phase, which correlated with spindle body assembly, kinetochore microtubule capture, and mitotic spindle elongation during anaphase A and B, visualized by fluorescence microscopy. Comparing synchronized and unsynchronized tubulin mutants of yeast cells, Pokorny´ et al. verified that synchronized yeast cells show more electric activity during M phase than non-synchronized yeasts. Direct measurements of electric resonant oscillations in microtubules have been presented at conferences by A. Bandyopadhyay. The technical aspects of direct detection of electric fields within a living cell have been discussed in a recent review. 

From our theoretical point of view, many of the unidentified forces regulating major cellular dynamic events during mitosis are probably electric forces generated by the synchronized oscillation of the electric dipoles within these super-macro organelle structures. 

The electrical properties of microtubules and centrosomes
 The electric field of the microtubule is generated by the synchronized oscillation of α and β tubulins. These tubulins form electric dipoles during microtubule polymerization; under intracellular energy excitation, synchronized oscillation of α and β tubulin subunits generates a longitudinal electric field around the microtubule  (Figure 1).



 Cifra et al. suggested that the source of the energy excitation could be hydrolysis of guanosine triphosphate (GTP) during the process of dynamic instability of microtubules, and also energy transferred from the movement of motor proteins or released from mitochondria as “wasted” energy from the citric acid cycle. We propose that the overall entropic environment within a living cell could be the source of energy for electric oscillation of microtubules. Cancer cells have different entropic states from normal cells as a result of the Warburg effect, which can cause mitochondrial malfunctions and further lead to alteration of cytoskeleton-based cellular elastoelectrical oscillations [27]. The microtubule networks of cancer cells generate an electromagnetic field with different frequencies. Thus, specific electromagnetic frequencies have been used to diagnose specific cancers [21,28], and tumor-specific modulating electromagnetic fields have been used to treat patients with advanced cancer with positive results [22,29]. The centriole of the centrosome is composed of α, β and γ tubulins organized differently from the subunits of microtubules; each centrosome comprises two centrioles, which are composed of nine triplets of microtubules. The two centrioles are arranged perpendicularly and surrounded by an amorphous mass of dense material (the pericentriolar material) [30]. As in microtubules, an electric field would be generated by synchronized oscillation between the α and β tubulins within the microtubule triplet of the centrioles (Figure 2).



Electric fields in centrosome separation and bipolar spindle body assembly 
Mechanisms of centrosome separation and bipolar spindle body assembly have been discussed in a recent review [31]. The process is still incompletely understood. Plus end-directed motor proteins such as kinesin 5 and minus end-directed motor proteins such as dynein are known to play dominant roles in centrosome separation and spindle assembly. However, centrosomal microtubules and microtubules of the nuclear envelope (NE) and cellular cortex need to move into close proximity for motor proteins to attach to both so they can generate the pulling forces. The current models assume a randomized mode of microtubule interaction, which is quite inefficient. For example, at a certain point a centrosome would have to stop moving until certain microtubules had grown sufficiently for appropriate bridging by motor proteins, particularly during prophase, when the centrosomes do not have many associated microtubules. When the electric fields of microtubules and centrosomes are considered, these structures are mutually attractive. Thus, centrosome movement along the microtubule networks of the cellular cortex and NE is more efficient. We can also envision a more autonomous mode of microtubule lattice formation within the cellular cortex and NE.

Centrosome Functions as a Molecular Dynamo in the Living Cell 2

Recent development in the field of quantum biology highlights that the intracellular electromagnetic field (EMF) of microtubules plays an important role in many fundamental cellular processes such as mitosis. Here I propose an intriguing hypothesis that centrosome functions as molecular dynamo to generate electric flow over the microtubules, leading to the electric excitation of microtubule EMF that is required for spindle body microtubule self-assembly. With the help of motors proteins within the centrosome, centrosome transforms the energy from ATP into intracellular EMF in the living cell that shapes the functions of microtubules. There will be a general impact for the cell biology field to understand the mechanistic function of centrosome for the first time in correlation with its structural features. This hypothesis can be tested with technics such as super resolution live cell microscope.

1. Introduction Centrosome was first discovered by Theodor Boveri in the 1880’s [1], it is the key organelle that is responsible for mitosis and meiosis in metazoan lineage of eukaryotic cells [2]. In animal cells, centrosome regulates the nucleation and spatial organization of microtubules, functioning as the primary microtubule-organizing center (MTOC) [3]. The centrosome is comprised of two centrioles that are surrounded by pericentriolar material (PCM). The two centrioles are perpendicularly arranged, one centriole has additional appendages at the end farthest from the other centriole (distal) and is called the mother or maternal centriole, the subdistal appendages of the maternal centriole also act like microtubule-anchoring sites [4] [5]. Pelletier et al. reported that subdiffraction imaging of centrosomes revealed pericentriolar material which had higher-order organizational features. Centrosome components adopt a toroidal pattern with progressively larger, overlapping diameters around the proximal end of the mother centriole in interphase cells. On one side, the toroid is slightly opened (gap) in the area where the daughter centriole is positioned [6], this higher order structural feature of centrosome may help the mother and daughter centriole to form an orthogonal configuration. In most of cases, each centriole is composed with 9 MT triplets and is ~0.5 μm in length and 0.2 μm in diameter [7] [8]. Recent studies in the field of quantum biology point to the possibility that electric magnetic interactions may involve in many fundamental cellular processes [9] [10]. In particular, the electromagnetic property of microtubule has been reported with both computation modelling and experimental evidences, Cifra et al. used computation model to simulate the electric pulse moving along microtubules, Bandyopadhyay et al. reported that nano sized electric pump was required for the self-assembly of microtubules in live cell simulator[11]-[13]. Medical treatment of cancer with cancer cell specific interfering EMF has been developed to disrupt the mitotic spindle microtubules of cancer cells [14]. Centriole produces an electromagnetic field apparently due to the longitudinal oscillation of its microtubules (MTs). Centrosome clustering is a hallmark of cancer cells. A cluster of centrioles is therefore presumed to produce an enhanced electromagnetic field. It is possible to target cancer cells using nano particles based on the enhanced electromagnetic property of cancer cells [15]. However, some important questions remain unanswered, what is the energy source of the intracellular electric field and what is the molecular mechanism that leads to the excitation of intracellular electric field? ATP is the most common cellular energy source. To transform the chemical energy in ATP into electric magnetic field within the living cell, cell needs to have a molecular dynamo to transform the mechanistic movement of protein complexes to directional movements of intracellular electrons, leading to the electric excitation of the spindle body microtubules as well as the M phase chromosomes, which is essential for mitosis [9].

2. Hypothesis 
Here I present a novel hypothesis that centrosome functions as a molecular dynamo in the living cell to generate electric current from the cytosol electrolyte to the spindle body and M phase chromosome, leads to the electric excitation of the spindle body and chromosome during mitosis. Based upon the structure of the centrosome, there is one microtubule in the center, and 9 microtubule triplicate outside, connected by motor proteins such as dynein and kinesin [3] 

https://vimeo.com/58347006

The mechanistic movement of these motor proteins will trigger the rotation of the microtubules triplets forming the barrel structure of the centriole to rotate around the center microtubule. The rotation and electric oscillation of each centriole will generate a dynamic electromagnetic field that mimic the physical structure of the centriole, and the orthogonal arrangement of centrioles of each centrosome will result in the microtubules of the barrel structure of each centriole to cut the electromagnetic field generated by the other centriole when rotating (Figure 1).



 Such a natural design makes centrosome to function as a molecular dynamo, generating directional electron flow through the dipolar structure of each individual microtubule in the centrosome, transforming the energy from ATP to electric current. During mitosis, centrosome is known to locate at the microtubule organizing center (MTOC), and only the mother centriole contains the sub-distal appendages that connect with spindle body microtubules, which allow the electrons to move from centrosome to the spindle body microtubules. During mitosis chromosomes are connected with spindle body microtubules with K-fibres, which are microtubule bundles that join kinetochores to the spindle poles. Pericentriolar material (PCM) is composed primarily of hyaluronic acid (HA) and has a similar negative charge density as DNA [16]. The electrons flow over the spindle body leads to the electric excitation of the spindle body and chromosome, generating an enhanced intracellular electromagnetic field during mitosis, which is consistent with the observation of yeast cell at M phase [17] [18]. Bandyopadhyay et al. reported nano-sized electromagnetic pumping is required for microtubule self-assembly [13], the electric excitation of spindle body microtubules is required for the self-assembly and growth of the spindle body microtubules during M phase. The basal body of cilium is a centrosome like cellular organelle [19], similar molecular mechanism is applied for the basal body centriole to generate electric flow over the cilium microtubules, forming the nano electromagnetic field that is required for the self-assembly of cilium microtubule. Collectively, my hypothesis proposes centrosome is at the center of the electric network that continuously drawn the cellular chemical energy from ATP to feed the intracellular electromagnetic field. 

On Centrioles, Microtubules, and Cellular Electromagnetism 3


1 Introduction 
Centrioles are tiny organelles lying adjacent to the nucleus of human and animal (eukaryotic) cells (see Fig. 1). Although their existence has been known for now over 100 years [1–7], centrioles have only recently been studied in any significant detail. This recent interest has been stimulated by: 

(1) dramatic advances in imaging technologies resulting in a better understanding of centriole geometry; 
(2) improved understanding of the important role of centrioles in cell mechanics—particularly in mitosis; and 
(3) the increasing belief that centriole malfunction occurs in virtually all solid tumor cancers. It is now known that centrioles have the following properties: 

(1) They have precise geometry, occurring in pairs as small annular cylinders (approximately 400 nm of long and 200 nm in diameter), with cylinder axes perpendicular to one another. 
(2) As with deoxyribonucleic acid (DNA) centrioles are selfduplicating and they duplicate at approximately the same time as DNA duplicates. 
(3) Centrioles are the only organelles without a membrane cover. 
(4) Centrioles are active participants in the cell division (mitosis) process. 
(5) During the S-phase of normal cell mitosis, each centriole duplicates once and only once. In abnormal mitosis, centrioles may duplicate multiple times and with disrupted geometry.



Figure 2 provides a drawing of a centriole pair. Interestingly, approximately 45 years ago, Dr. Paul Schafer, while working at the Veterans Administration Hospital in Washington, D.C., discovered and reported  that esophageal cancer cells have distorted centriole geometries. He suggested that cancers develop due to abnormalities in the electromagnetic fields surrounding the centrioles. Unfortunately, as with Boveri, Schafer’s work has received relatively little attention until recently. The following paragraphs summarize recent and state-of-the-art findings about centrioles, their inner workings, and their overall effect upon cell electropolarity. How these findings could be an aid in cell research is also discussed—particularly in solving the problem of in vivo insertion of therapeutic nanoparticles .



2 Centriole Geometry
Figure 1 provides a typical drawing of a cell interior. The centrioles are seen as the small perpendicular organelles adjacent to the nucleus. In Fig. 2, we have a closer view of the centriole pair. long as the mother. The daughter centriole is attached to the base, or proximal, end of the mother centriole. In this configuration, the axis of the daughter intersects the axis of the mother, with the axes of the two centrioles being perpendicular. Surrounding the centriole pair, at the adjoining base, is an amorphous cloud of numerous proteins known as the MT organizing center (MTOC) . The centrioles together with the MTOC are known as the centrosome. The centrosome, being roughly spherical, has a diameter of approximately 4 lm. The MTOC is believed to be “electron dense” . In this regard, with the MTOC at the intersecting bases of the centriole
pair, the proximal or base end of the centriole is given negative polarity or potential. The distal ends are thus positive.

3 Centriole Duplication and Mitosis
 Centrioles are the principal drivers of cell division and duplication (mitosis). Mitosis is typically described as occurring in four phases: In the first phase (the “prophase”), each centriole becomes a new “mother” centriole supporting the creation of a new daughter centriole on its side and at its proximal end. As these daughter centrioles develop and grow, the original centriole pair becomes two pair, with the original mother centriole having a new daughter, and the original daughter having a new daughter of its own .



Figure 5 illustrates the two-pair configuration. 

During the time that the centrioles are duplicating, the nucleus membrane begins to soften, break down, and the DNA condenses in preparation for division. Finally, the connection between the centriole pairs stretches and the pairs move apart. Interestingly, the original mother centriole with its new daughter remains somewhat stationary while the less mature daughter-new daughter centriole pair moves away, around the nucleus to the opposite side. During the movement, this immature centriole pair becomes mature. Even though the centriole pairs are separating, they remain connected by MTs about the collapsing nucleus—almost in the shape of a football. Taken as a group, these MTs are known as the “mitotic spindle” (see Fig. 6). 



The remaining three phases (metaphase, anaphase, and teleophase) complete the cell separation process with the metaphase being the creation of a symmetric structure between the two pairs of centrioles; the anaphase being the separation in the midplane; and finally, the teleophase being the formation of new nuclear membranes about the separated parts. The two centriole pairs each align themselves adjacent to the separated nuclei respectively. The separated nuclii then move further apart, each taking with it approximately half of the original cell organelles and half of the remaining cell material (the cytoplasm). Thus the original cell becomes two cells.

4 Cell Electropolarity via Longitudinal MT Vibration 
Numerous studies have shown that the MTs within a centriole generate an electromagnetic field about the centriole. This field is believed to occur due to longitudinal (axial) vibration of the MT filaments. Consider again Fig. 3 showing again a sketch of an MT, (1 of 27), parallel to the axis of the centriole barrel. The proximal (or base) end of the MT is immersed in the electron dense material of the MTOC. With this proximal end being negative, and thus the distal end being positive, this positive distal end is immersed in the cloud of material of the centrosome, or pericentriolar material. As noted earlier, the MT filaments are composed of alternate a-tubulin and b-tubulin protein pairs (“dimers”). A single dimer has a high electric charge difference (polarity) along its axis. Then with the filament dimers being arranged in a series of dimers along the filament length, there is a voltage change from the proximal (negative) to the distal (positive) end of the filament. It is the tubulin that is the source of this potential difference. An MT filament surface is relatively smooth, allowing for relatively free longitudinal, oscillatory movement (vibration) of the filament. Then taken together the longitudinal, or axial, vibration of the 13 filaments of an MT and then the 27 MTs making up the centriole barrel produce the electromagnetic field surrounding the centriole . Interestingly, this field is also found to be ferromagnetic. Also of interest, the fundamental vibration frequency of an MT filament is approximately 465 MHz, although this frequency is continually changing due to the ongoing length changing of the filaments. The electropolarity of the centrioles enables them to exert forces at a distance—that is, forces without physical contact. Also, it is this high electropolarity of the centriole, lying next to the nucleus, which produces the overall electropolarity of the cell. The transepithelial potentials, that is, the potential difference across a cell membrane cover may range from a few millivolts to tens of millivolts. Finally, as one would expect, the peak of cell electropolarity occurs during mitosis, when there are four, instead of two, centrioles. Correspondingly, the MTs then orient the mitotic spindle along the axis of cell polarity.

Findings 
(1) Electromagnetics play an important role in cell functioning and especially in cell duplication and division (mitosis). 
(2) Cell electropolarity arises from the centrioles via the MTs of the centriole blades. 
(3) The MTs obtain their polarity via a, b dimers arranged in series along the filaments of the MT wall. 
(4) Overall cell electropolarity is increased during mitosis due to the presence of two pairs of centrioles. 
(5) Overall cell electropolarity is even greater in cancer cells due to the presence of supernumerary centrioles and centriole clustering. Indeed, supernumerary centrioles tend to cluster together—apparently due to electromagnetic attraction [70]. 
(6) Clinically, it is found that breast cancer tissue has sufficiently large electrical polarity that it can be detected relative to surrounding tissue at the breast surface, and the aggressiveness of breast cancer is proportional to the degree of centrosome amplification.
(7) In general, there is a small but steady extracellular voltage gradient between cancer tissue and neighboring normal tissue . 
(8  Centrosomal amplification is regarded as a “hallmark” of cancer cells —that is, all major cancer cells have centrosomal amplification . 
(9) In an early stage of mitosis, electromagnetic forces send the less mature pair of centrioles around the nucleus to the other side. 
(10) The electropolarity associated with centriole clustering may be useful for tumor identification and treatment using charged nanoparticles

Open Questions and Needed Data 
(1) Why is centriole geometry so precise and why does it have the particular form that it has? 
(2) During centriole duplication, why does the daughter centriole arise and develop on the side and at the base of the mother centriole? 
(3) What is the magnitude of the electropolarity change along a centriole length, across a centriole pair, and across a duplicating pair of centrioles? 
(4) What is the magnitude of the electropolarity change across a centriole cluster of a cancer cell? 
(5) Is the electropolarity of a centriole cluster sufficiently strong to attract charged nanoparticles? 
(6) What is the range of frequencies of the longitudinal vibrations of MTs with variable lengths? 
(7) What is the three-dimensional map of potential of a normal cell? Additional experimentation will be required to answer these questions. To this end, it now appears that the best approach is to use current nanotechnology including the use of atomic force microscopy with magnetic sensitive probing tips. This is a subject of ongoing local research.

1)http://download.springer.com/static/pdf/238/art%253A10.1186%252F1742-4682-9-26.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1186%2F1742-4682-9-26&token2=exp=1464118570~acl=%2Fstatic%2Fpdf%2F238%2Fart%25253A10.1186%25252F1742-4682-9-26.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1186%252F1742-4682-9-26*~hmac=84a38aee1ee3fcaf89567ff0515b79c6436aad91a7cf73d89a1a084def147165
2) http://file.scirp.org/pdf/ABB_2015072015020110.pdf
3) file:///D:/Downloads/nano_005_03_031003.pdf

Centriole Evolution 1

Summary

Centrioles are cylindrical structures found at the core of the mitotic spindle pole, which also act as basal bodies to nucleate formation of cilia. Centrioles have a complex, nine-fold symmetric structure, and reproduce by an intriguing duplication process. The complexity and apparent self-reproduction of centrioles raises the question of how such a structure could have evolved, making them a favorite topic for theological speculation by "intelligent design" creationists. In fact, centrioles are capable of robust self-assembly and can tolerate dramatic perturbations while still maintaining basic functionality. Far from being irreducibly complex, centrioles appear to be based on a rather minimal underlying core structure requiring only a handful of genes to construct.

Introduction: phylogeny, terminology, and theology

The centrosome (1) is a bipartite structure, consisting of a pair of cylindrical microtubule-based organelles called centrioles (2), embedded in an amorphous network of proteins known collectively as Pericentriolar Material (PCM). The microtubule-nucleating function of the centrosome is carried out by gamma tubulin ring complexes docked on the PCM. In contrast to the PCM, which has relatively little discernable structure (3), centrioles have a remarkably complex structure (4), which has raised the question of how something so complicated may have evolved. The complexity of the centriole suggests that a large number of genes may be required to build it, which poses a challenge for evolution because lack of any one of those genes would eliminate the functionality of the centriole, so that a fitness benefit would only be accrued once the entire gene set was established. This review will address the question of centriole evolution in light of recent experimental results which suggest that centrioles, while complicated-looking, may be substantially less complex than previously suspected.

Centriole structure and function

Centrioles consist of nine microtubule triplet blades arranged in a cylinder. At one end of the centriole is a spoke-like arrangement called the cartwheel. A variety of fibers and protrusions extend from the centriole and probably act to anchor it to various elements of the cytoskeleton (5,6). What is all this complex structure for, given that the main microtubule nucleating activity of the centrosome resides in the PCM and not the centriole? Experimental evidence shows that centrioles are required for the formation of a persistent centrosome (7) but when centrioles are removed from cells, bipolar spindles can still form using an alternative self-organization pathway that does not require centrosomes (8). What then is the real function of centrioles, if they aren't needed for mitosis? A look at the phylogeny of eukaryotes (9) shows us the answer (Figure 1).




Presence of centrioles correlates strictly with presence of cilia throughout eukaryotic phylogeny. 
(+) and (−) indicate whether centrioles or cilia are present or absent, respectively, in the given phylum. All phyla that currently lack centrioles appear to have descended from ancestors that once contained centrioles.

All species, without exception, that have centrioles, always have cilia at some stage of their life cycle, and vice versa. For example, lower plants such as mosses and ferns lack both centrioles and cilia in most cells but suddenly form them both during spermatogenesis. Indeed, centrioles are strictly required for the formation of cilia, and perform multiple functions on the behalf of cilia (10). First of all, the microtubule doublets of cilia grow as a direct outgrowth of the microtubule triplets of the centriole (11,12). When a centriole forms a cilium, it becomes known as a Basal Body. In addition to directly nucleating ciliary microtubules, basal bodies dictate the orientation and positioning of cilia (13), and act as recruiting centers for molecules involved in ciliogenesis (14). The evolution of centrioles must thus be considered together with the evolution of cilia (15,16), and any fitness benefit that a eukaryotic precursor organism could have attained from the innovation of centrioles must have come from the ability to make cilia.

Self-reproduction of centrioles?

In most cells, centrioles form by an apparent "duplication" process by which each pre-existing centriole gives rise to a new centriole, at right angles to the first one (17). When this happens, the old centriole is called the mother and the new one the daughter. Microsurgical removal of centrioles indicated that cells lacking centrioles cannot form new ones, suggesting that the duplication pathway was the only way that centrioles could form (18). If new centrioles can only form from old ones, how could centrioles ever evolve in the first place within a cell that initially lacked them?
This type of chicken-and-egg paradox was heightened by claims that centrioles contained their own organellar genome (19,20). Indeed, if centrioles contained their own genomes, it would strongly imply that they arose via an endosymbiotic mechanism similar to that which gave rise to mitochondria and chloroplasts (21). Alternatively, it has been proposed that centrioles may have evolved from a virus (16). Requirement of an internal genome would also explain why centrioles undergo duplication and would also potentially make it impossible for centrioles to form de novo. However, a substantial number of experiments have subsequently proven beyond any doubt that centrioles do not contain a DNA-based genome of their own (22–26). It has been clearly shown that centrosomes contain specific RNA molecules associated with them, but these are encoded by genes that, while having an unusual intron-poor structure, are still found within the nucleus (27).

Since centrioles apparently do not contain their own genomes after all, this removes one part of the chicken and egg paradox. The problem posed by centriole duplication is also easily disposed of because, in fact, centrioles can form de novo. De novo formation is a natural occurrence in certain organisms that lack centrioles through parts of their life cycle and then re-acquire them at other stages (28,29). When centrioles are removed even from "normal" cells, new centrioles can form de novo with great efficiency (30,31). Why, then, did initial surgical studies suggest centriole duplication was obligatory? One possibility is the cells were damaged during the procedure. However it has also been shown that experimental removal of centrioles results in cell cycle arrest (32) in G1. De novo centriole formation can only occur in S phase (30,31), so G1 arrest would prevent centriole formation. This G1 arrest appears to be due to an increase in stress-sensitivity, and if the stress response is circumvented, de novo centriole assembly can occur (33).
Thus, the apparent paradox raised by centriole duplication turns out to be a non-issue. Centrioles can form de novo in cells that lack them without any difficulty, thus providing a way for the whole centriolar chain of being to begin.

Centriole complexity is not so irreducible after all

Centrioles look highly complex, but so do snowflakes. Extremely simple self-organizing chemical and physical processes can generate structures of extraordinary complexity, and one must be careful to distinguish between two different types of complexity. One type of complexity is called "informational complexity" which can be measured as the number of bits of information required to explicitly describe the structure in question. For instance, one could recognize image features such as corners and edges and record their positions within the object. Structures that look visually complicated, such as a Persian rug, will have much higher informational complexity than a simple-looking pattern such as a checkerboard. An alternative measure of complexity stems from the fact that simple computer programs can generate complex fractal patterns. Depending on the pattern, a very simple program may suffice, while for other structures, it may take a longer program to generate the pattern. This type of consideration has led to a newer way to describe structures in terms of their "algorithmic complexity", also known as Kolmogorov complexity, which is the size of the smallest computer program (usually measured in bits) sufficient to generate the pattern (34,35). Evolution and genomics is concerned with algorithmic complexity and not informational complexity. No matter how visually complicated a centriole is, its evolvability depends only on how complicated the genomic "program" must be that generates the structure.

We must therefore consider how many different genes are really needed to build a structure that is minimally functional as a centriole. This question has two parts: (a) how much of the structure of the centriole is really critical for its function, and (b) how many genes are necessary to generate the critical core structure. We will tackle the first question first - how much could the structure of a centriole be altered and still work? In this case, "work" would be defined as being able to provide at least some fitness benefit to a cell. Since the main job of centrioles is to form cilia, we can focus on how centriole structure contributes to assembly of cilia. Templating of cilia by centrioles requires is a set of preexisting microtubules, but the canonical arrangement of nine triplets in the centriole and nine doublets in the axoneme is by no means absolutely required for ciliary function, as organisms are known that have other numbers of centriolar or axonemal microtubules (36–39). These cilia and flagella are motile, despite deviating from the canonical ninefold symmetry, hence we can only conclude that there is nothing magical about the number nine. Moreover, it is possible for centrioles having nine triplets to generate cilia having more than nine doublets, indicating that cilia have a degree of self-organization independent of the influence of centrioles (40). Mutants in proteins of the centriole cartwheel can produce centrioles with variable numbers of triplets instead of nine (41,42), and these modified centrioles can still form cilia, demonstrating that ninefold symmetry is not an essential feature of centrioles. It is also important to point out that the existence of a symmetrical array of triplets (be it nine-fold symmetric or with some other symmetry) is not required for the triplets themselves to form. Mutants also exist in which centrioles form asymmetric arrangements of triplet-containing units in variable orientations (43). These studies demonstrate that single triplet subunit-sized chunks of centrioles can form without the overall rotational symmetry being present, so that if a single chunk could perform some useful function, this could be selected for prior to the development of the final nine-fold symmetrical structure.

Although ciliary motility is a highly coordinated process that might be quite sensitive to deviation away from nine-fold symmetry, cilia also play important sensory functions that would require little more than the microtubules and a membrane into which receptors could be localized. Cilia can also drive gliding motility which is independent of normal ciliary motility and might not require strong nine-fold symmetry (44). In one interesting protist, the cilium consists almost entirely of an elongated central pair of microtubules, lacking the nine doublets over most of its length, yet this is able to drive swimming (45). It thus seems reasonable that even a very rudimentary centriole-like precursor could allow formation of a proto-cilium that would give cells a tremendous advantage in terms of either sensory or gliding functions.
The second key question is how many genes would have to evolve in order to generate a centriole. One way to get at this question is to ask how many genes are essential to maintain centrioles. A recent genome-wide RNAi screen in Drosophila has argued that only nine genes are required for centriole duplication (46). The apparent complexity of the centriole proteome, which likely consists of at least 50–100 proteins (47,48), does not contradict the idea that only a small core set of genes are essential for centriole formation, provided the majority of the centriole proteins constitute add-ons, for example fibers that attach centrioles to different cytoskeletal elements. Combining these considerations, it is apparent that only a small number of molecular innovations would be needed to produce some reduced, fragmentary version of a centriole that could in turn nucleate some sort of microtubule-based cellular extension that would be useful for gliding or sensation.

I propose that the original evolutionary precursor to the centriole may have resembled a single triplet blade subunit of the present day centriole, and consisted of a microtubule doublet or triplet structure that was able to extend a rigid proto-cilium consisting of a single doublet surrounded by plasma membrane out into the extracellular environment (Figure 2). 





Proposed evolution of a proto-centriole capable of nucleating a primitive cilium-like structure. 
(A) Evolution of tektins allows formation of stable microtubule doublet and triplet structures. 
(B) Acquisition of one or more appendage proteins allows docking of proto-centriole onto cell cortex. 
(C) Microtubules can extend from the end of the double structure. 
(D) If microtubules extend from the end of a docked doublet it would produce a primitive cilium-like structure that could be used for sensory or gliding functions.


Extension of microtubules from the end of the doublet would not require additional evolutionary novelty since it is known that centriole triplets can serve as templates from assembly of purified tubulin (12). The resulting structure, while simple, would have been able to provide basic sensory and gliding motility functions. The molecular requirements for formation of such a structure could be quite minimal: one or more proteins involved in forming the doublet microtubule structure plus a protein capable of linking the base of the structure to the cellular cortex. It is not currently known how microtubule doublets and triplets form, but the tektin family of proteins is thought to be involved in the process (49). Evolution of one or more tektins, plus one or more proteins that can bind microtubule doublets and attach to the cortex, such as ODF2/cenexin (50), might have been sufficient to produce a centriole precursor with basic function.

Once the original proto-centriole was established, it eventually was modified by addition of further gene products to produce the characteristic nine-fold symmetric array of triplets found today. Genes required for this transition would be recognizable as those which when mutated lead to aberrations in symmetry without affecting assembly of the triplet blade subunits themselves. This phenotype has been seen for mutants in the centriole cartwheel proteins SAS-6 and BLD10 (41, 42) suggesting that these genes may have evolved after the proto-centriole in order to bring about the modern cylindrical symmetric structure. The acquisition of a symmetric cylindrical arrangement might provide a structure with greater mechanical strength to support more powerful motility, and might allow a cilium to project at a right angle to the cell surface by presenting a rotationally symmetric array of attachment appendages. Although as discussed above variations in structure are seen in some lineages, the cylinder of nine triplets is by far the most common arrangement. This relatively invariant centriole architectural plan seen among eukaryotes, combined with the fact that the ninefold triplet architecture is found even in the earliest branching eukaryotic lineages, such as Giardia, suggests that the structure evolved just once and was then inherited throughout the eukaryotes. Modifications to centriole structure, including the reduction of centrioles to discs of singlets in nematodes would thus have occurred by secondary loss events.


De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly 2

Centriole biogenesis does not strictly depend on SAS-6 self-assembly, and may require preexisting centrioles to ensure structural accuracy, fundamentally deviating from the current paradigm.
Cells pass on their characteristics or “traits” to new generations in the form of DNA molecules. DNA provides the instructions to make proteins, which may then assemble into larger structures without using any external templates in a process called self-assembly. However, when a cell divides, DNA is not the only element that is passed on to the daughter cells; many large protein structures that have assembled in mother cells are also divided between the daughter cells. The daughter cells may then produce extra copies of these protein structures, but it is not known whether the pre-existing structures are involved in this process.
Centrioles are complex structures made of proteins and play a crucial role in cell division. One of the main components of centrioles is a protein called SAS-6. Recent studies have shown that SAS-6 molecules can bind to each other to form “oligomers”. This process, which is called self-oligomerization, has been proposed to drive the formation of centrioles.
Now, Wang et al. examine whether centrioles can form properly in cells when no other centrioles are present. The experiments show that centrioles can indeed form, but they are prone to structural errors. In contrast, centrioles that form in the presence of older centrioles are essentially free of errors. The experiments used human eye cells that were missing the gene that encodes SAS-6. These cells could not make centrioles, but when SAS-6 was re-introduced into these cells, new centrioles formed. Unexpectedly, re-introducing a mutant form of SAS-6 that cannot form oligomers into the cells still allowed new centrioles to form, which shows that self-oligomerization of SAS-6 is not essential for the assembly of centrioles.
Together, Wang et al.’s findings challenge the idea that SAS-6 self-oligomerization is involved in the formation of centrioles, and suggest that preexisting centrioles may help to minimize errors in the formation of new centrioles.

Introduction

Centrioles are microtubule-based, ninefold symmetrical structures essential for centrosome and cilia formation. In cycling cells, centrioles are maintained in fixed numbers, and formed through canonical duplication depending on pre-existing (or mother) centrioles. In the absence of pre-existing centrioles, however, de novo synthesis can occur (Khodjakov et al., 2002). The number of centrioles formed through the de novo pathway is highly variable (Khodjakov et al., 2002; La Terra et al., 2005), providing an explanation for why canonical duplication dominates in dividing cells. In contrast to cycling cells, in post-mitotic cells such as multi-ciliated epithelia, the genes required for centriole assembly are highly up-regulated (Hoh et al., 2012) to produce large, variable numbers of centrioles prior to ciliogenesis, a process thought to primarily depend on de novo assembly (Dirksen, 1991). Interestingly, a recent study showed that the production of high quantities of centrioles in mouse multi-ciliated epithelia is in fact driven by the pre-existing centriole rather than through de novo assembly (Al Jord et al., 2014), suggesting that the presence of pre-existing centrioles may have additional roles other than the number control for centriole biogenesis.
Centriole biogenesis, canonical or de novo, starts with cartwheel assembly, a geometric scaffold that defines the shape and structural integrity of centrioles (Anderson and Brenner, 1971). The backbone of the cartwheel is characterized by a central hub from which nine spokes emanate (Anderson and Brenner, 1971) and is primarily made of the centriolar protein SAS-6 (Kitagawa et al., 2011; van Breugel et al., 2011). SAS-6 exists as dimers, which can self-oligomerize in vitro via an N-terminal head domain, forming a ring resembling the central hub, and C-terminal tails pointing outwards as spokes (Kitagawa et al., 2011; van Breugel et al., 2011; van Breugel et al., 2014), albeit not always ninefold symmetric in vitro(Cottee et al., 2011). Nevertheless, these elegant discoveries raise an exciting proposal that the self-assembly property associated with the N terminus of SAS-6 drives cartwheel and centriole formation.

In contrast to the SAS-6 self-assembly model, a template-based assembly model, dependent on the interaction of the C-terminal tail of SAS-6 with the lumen of mother centrioles, has recently been proposed to initiate canonical duplication (Fong et al., 2014). During S phase, SAS-6 molecules are first recruited to the proximal lumen of the mother centriole prior to centriole duplication, adopting a cartwheel-like organization through interactions with the luminal wall, rather than via their self-oligomerization activity. This leads to a proposal that mother centrioles may function as the template to shape SAS-6 assembly, thereby preserving the geometric shape of the centriole that otherwise cannot be ensured by SAS-6 self-assembly alone. Notably, the template-based model appears incompatible with de novo centriole synthesis in which no pre-existing centrioles are required. However, as the nature of de novo synthesis, for example, whether it is indeed based on SAS-6 self-assembly, has not been determined, it is premature to accept or reject any of these ideas.

Exploring the evolutionary history of centrosomes 3

1. Introduction

Centrosomes are membrane-free organelles that serve as main microtubule-organizing centres in distinct eukaryotic lineages. Through their ability to organize microtubules, they are involved in cell polarity and cell division, and play key roles in the development of most animal species [1,2]. In animal cells, the centrosome is composed of two centrioles surrounded by the pericentriolar material (PCM). The PCM has a precise organization, which derives from the hierarchical recruitment of a small number of large coiled-coil proteins around the centrioles [3,4]. The centrioles ensure the stability and the duplication of the centrosome, whereas the PCM anchors microtubule-nucleating complexes and cell cycle regulators. Centrosome duplication occurs by the assembly of two new centrioles in the immediate vicinity of the pre-existing centrioles, and a pair of centrioles is inherited by each daughter cell following mitosis. The timing of centrosome duplication may vary from one species to another, but it is always precisely coupled to cell cycle progression [5–9].

The centrosome can also migrate to the cell periphery, where the older centriole, called the mother centriole, can dock at the plasma membrane and nucleate the assembly of a cilium. Most mammalian cell types assemble a primary cilium in G1 or G0, which is typically a non-motile cilium involved in sensory functions such as cell–cell signalling or flow sensing [10–13]. Non-motile cilia are also critical for the function of sensory neurons in a range of animal species, including photoreceptors as well as olfactory, mechanosensory or chemosensory neurons [14–16]. In other cell types, the mother centriole can template the assembly of a motile cilium or flagellum, for instance, in the zebrafish kidney or in most sperm cells [17–19]. In this configuration, the mother centriole is functionally equivalent to the basal bodies that nucleate cilia or flagella in a range of unicellular eukaryotes. Centrioles and basal bodies are highly similar at the ultrastructural level, and indeed the key factors for centriole assembly are conserved in the genomes of all ciliated organisms [20–24].  The ancestral function of centrioles was likely to nucleate cilia, as evidenced by the co-distribution of centrioles and cilia across the eukaryotic tree [25]. Cilia are involved in locomotion through either beating or gliding motility, and in addition have sensory functions in diverse eukaryotes, suggesting an ancient association between motion and sensory perception [26,27].


In contrast to basal bodies, centrosomes are by definition central organelles. The term centrosome (etymologically, central body) is a generic term to design any isolable single-copy organelle having in common three basic properties: 


(i) to generally maintain itself at the cell centre due to its microtubule-nucleating/anchoring activity, 
(ii) to duplicate once during the cell cycle and 
(iii) to be physically associated with the nucleus [26]. 


The fact that the centrosome and the basal body complex can interconvert in many types of animal cells suggests that the centrosome evolved by internalization of the basal body complex. At which point during evolution and in how many different lineages this happened is not clear, however. Besides animals, organisms with life cycle stages where cilia are absent and internalized centrioles organize the microtubule cytoskeleton include brown algae and Amoebozoa such as Physarum polycephalum [28]. In addition, some lineages completely lost centrioles and cilia during evolution but assemble organelles that are functionally equivalent to animal centrosomes. These include higher fungi, Amoebozoa such as Dictyostelium discoideum, diatoms and probably Ichthyosporea. The centrosomes of higher fungi are called spindle pole bodies (SPB). In Saccharomyces cerevisiae, the SPB is a multilayered cylindrical organelle embedded within the nuclear envelope [29]. The outer layer connects cytoplasmic microtubules, whereas the inner layer organizes spindle microtubules during mitosis, which occurs without disruption of the nuclear envelope. During cell division, a new SPB is assembled on a structure attached to the old SPB called the half-bridge [30]. The centrosome in the fission yeast Schizosaccharomyces pombe has a less obvious internal structure than its budding yeast counterpart, and is embedded in the nuclear envelope only during mitosis. It duplicates through the formation of a half-bridge similar in its structure and molecular composition to S. cerevisiae half-bridge, however [31]. In Amoebozoa such as D. discoideum, the centrosome is called a nuclear-associated body (NAB). The NAB consists of a match-boxed shaped three-layered core surrounded by a corona of amorphous material. Microtubules are organized from the corona, which is thus regarded as equivalent to the PCM of animal centrosomes. Duplication of the NAB occurs in a very unusual way: early in mitosis, the central layer disappears, and the two outer layers peel apart and separate to organize the mitotic spindle. At the end of telophase, the layers fold in two and a central layer and a corona are reformed [32,33]. In diatoms, the microtubule centre (MC) is closely associated with the interphase nucleus and the Golgi apparatus. The MC itself does not duplicate, but it is closely associated to a cubic laminar structure called the polar complex, which forms prior to mitosis and splits into two ‘polar plates’ that form the poles of the mitotic spindle [34]. Finally, Ichthyosporea (also called Mesomycetozoa) assemble spindle pole bodies reminiscent of the SPB of higher fungi [35,36]. Ichthosporean SPBs are plaque-like structures apposed to the nuclear envelope in close proximity to the Golgi apparatus. By electron microscopy, interphase cells have a single SPB organizing cytoplasmic microtubules and cells preparing for mitosis have two SPBs, one at each end of the nucleus [35]. The SPB cycle has not been studied in these organisms however, and it is not known how duplication occurs.

Despite this great diversity of shape, acentriolar centrosomes in fungi and Dictyostelium share some components with animal centrosomes, which suggest a common evolutionary origin [37–41]. In this paper, we will explore the evolutionary history of centrosomes by comparing microtubule cytoskeleton ultrastructure and centrosome composition in different eukaryotic lineages in the light of recent phylogenetic studies. We will attempt to bring insights into whether these centrosomes derive from an ancestral centrosome or evolved independently from the flagellar apparatus of distinct unicellular ancestors. This is an important point as it can affect the way we understand the similarities and differences between widespread model organisms. Because ultrastructural and molecular data are otherwise limited, we will focus on the lineage comprising Dictyostelium, higher fungi and animals, called Amorphea. Finally, we will discuss the evolution of centrosome function in animals.

6. Concluding remarks

the  parsimonious hypothesis at this point is that these centrosomes result from convergent evolution. The main argument in favour of this second scenario is the phylogenetic position of Apusozoa. These excavate-like flagellates are more closely related to opisthokonts than to Amebozoa such as Dictyostelium. This suggests that the common ancestor of opisthokonts and Apusozoa was itself an excavate-like flagellate, and that opisthokonts and Amoebozoa evolved independently from the flagellar apparatus of distinct ancestors. Some key centrosome properties derive from properties of the ancestral basal body complex, such as duplication once per cell cycle, connection to the nucleus and role in organizing cytoplasmic microtubules. It is thus plausible that these characteristics were retained independently during evolution of the different centrosomes.

we now have a better picture of the ancestral eukaryotic cytoskeleton, and it turns out to be surprisingly complex. The last common ancestor of all eukaryotes was probably a biflagellate with a very elaborated microtubule cytoskeleton, an architecture that is still seen in many living species but was simplified in the evolution of many others [61,62]. The difficulty when trying to reconstitute the evolution of cellular architectures is that, due to a limited fossil record, we can only guess from the observation of living species what the sequence of events was. 

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835302/
2) https://elifesciences.org/content/4/e10586
3) http://rstb.royalsocietypublishing.org/content/369/1650/20130453

Centrosomes back in the limelight 1

As in all fields, but especially in ones that span more than a century, during which conceptual frameworks and experimental approaches have changed substantially, there is a need to ensure some shared basic terminology to facilitate communication between members of the community and accelerate entry into the field for newcomers. For instance, until when should a procentriole be referred as such before being called a centriole in the canonical centrosome duplication cycle? Hereafter, we use the term procentriole to refer to a centriolar cylinder from the moment it is discernible next to the proximal end of a parental centriole, approximately at the G1/S transition, until mitosis of that cell cycle (figure 1a). During this time interval, a procentriole and the parental centriole next to which it emerges are referred to collectively as a diplosome. After disengagement of the procentriole from the parental centriole during mitosis and subsequent entry into G1, the younger structure, which used to be the procentriole, is now referred to as the daughter centriole, and the older one as the mother centriole (figure 1a). The mother centriole harbours distal and sub-distal appendages whose distribution mirrors the ninefold symmetry of the centriole, and which are acquired at the end of the cell cycle following that in which the procentriole emerged. The distal appendages mediate docking of the mother centriole to the plasma membrane in cells that exit the cell cycle. Once docked in that location, the mother centriole is referred to as the basal body, and by some workers as the kinetosome [7], and serves to template formation of the axoneme in cilia and flagella. Note that nowadays the basal body is frequently referred to as a centriole, both for simplicity and because basal bodies and centrioles can interconvert in many cell types. Note also that often, including in the present piece, the plural ‘centrioles’ is used to refer indiscriminately to all centriolar cylinders (i.e. jointly to centrioles and procentrioles).




Centrosomes in human cells. 
(a) Representation of a pair of centrosomes in human cells viewed from the side during the S phase of the cell cycle. The lines designated 1, 2 and 3 indicate the positions corresponding to the cross sections shown in 
(b). The parental centrioles are approximately 450 nm long and approximately 250 nm in outer diameter. The grey region in the distal part represents the filled lumen in the region where centrin concentrates [6]. Note that for simplicity the cartwheel is represented with only four slices and that it is present only in the procentriole in human cells. Similarly for simplicity, the PCM/centrosomal matrix is represented solely around the proximal region, even though it is also present to a lesser extent around the more distal segments. (b) Corresponding cross sections, viewed from the proximal end, in regions 1 (proximal part of the centriole, with cartwheel highlighted and triplet microtubules denoted A, B, C), 2 (central part of the mother centriole, also with triplet microtubules) and 3 (distal part of the mother centriole, with double microtubules denoted A, B). (Online version in colour.)

Apart from centrioles, another main character in the plot is the pericentriolar material (PCM), also known as the centrosomal matrix, an electron-dense region that surrounds the centriolar cylinders, particularly their proximal part, and together with them constitutes the centrosome (figure 1a). It is now clear that centrioles and PCM are intimately linked to fulfil the numerous functions of the centrosome. However, when the centrosome was equated to an MTOC—when microtubule nucleation was the main function envisaged for the entire organelle—the dominant view was that centrioles were not important for centrosome function. That centrioles are instrumental in maintaining centrosome integrity was demonstrated in human cells by injection of monoclonal antibodies against polyglutamylated tubulin, a post-translational modification of α- and β-tubulin particularly prevalent in centrioles [8]; see §5). This led to centriole loss and subsequent dissolution of the entire centrosome. In Caenorhabditis elegans, partial depletion of centriolar components by RNAi results in smaller centrioles that recruit less PCM than in the wild-type, further indicating that PCM-size scales with centriolar material [9,10] . Therefore, centrioles play a fundamental role in assembling the centrosome organelle.
There are other cast members that are neither centriolar nor PCM components, yet clearly important for the overall architecture of the centrosome (figure 1a). These include the inter-centriolar linker that connects the mother centriole and the daughter centriole in G1, as well as the two diplosomes thereafter, as well as centriolar satellites, granules approximately 100 nm in diameter that remain incompletely described with respect to their composition and function, apart from being important for primary cilium assembly [11,12].
Besides knowing the cast of characters, it is also important to ensure that the nomenclature of the molecular players that participate in the play is accessible to a broad base of scientists. Too many proteins have been referred to under more than one name. For instance, the human protein related to C. elegans SAS-4 (Spindle ASsembly abnormal 4) has been referred to as SAS4 (to indicate its relatedness with the worm protein), as CPAP (for Centrosomal P4.1-Associated Protein, as it was first named, before the relationship to SAS-4 was known) or CENPJ (for Centromere Protein J, for reasons that remain unclear). Although this naming plethora is not an issue specific to the centrosome field, a concerted effort would be welcome to clarify the language.

The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries 1

Abstract

Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes.

Centrosomes and associated components determine the geometry of microtubule arrays throughout the cell cycle, and thus influence cell shape, polarity and motility, as well as spindle formation, chromosome segregation and cell division1. Importantly, centrioles also function as basal bodies for the formation of cilia and flagella. These in turn play important roles in locomotion, transport and signalling2. Phylogenetic studies indicate that centrioles/basal bodies existed in the last common ancestor of eukaryotes but were lost from specific branches, such as yeasts and vascular plants3. Their presence correlates strictly with the occurrence of cilia, indicating that selective pressure was exerted on basal body functionality. Aberrations in centriole/basal body formation and function are associated with a plethora of human diseases, including ciliopathies, brain diseases and cancer. Accordingly, recent years have seen a surge of interest in the biogenesis and function of these elaborate intracellular structures, as reflected in the number of excellent and comprehensive reviews now published4–8. Here we focus on recent advances and a selection of seminal papers that bear on centriole biogenesis, duplication, function and association with cellular asymmetries.

Centriole biogenesis and the control of centriole number

In cycling cells, exactly one new centriole forms adjacent to each pre-existing centriole, reminiscent of the replication of DNA. In contrast, in differentiating multiciliated epithelial cells, hundreds of centrioles are formed near-simultaneously adjacent to deuterosomes, amorphous proteinaceous structures unique to this cell type. Importantly, it is now recognized that ‘de novo’ formation of centrioles occurs more commonly than previously thought9 and even cycling cells display this ability if the resident centrioles are experimentally removed10. Moreover, recent studies demonstrate that the pathways underlying centriole/basal body formation in eukaryotes share a common set of key regulatory proteins, indicating that they represent variations on a common theme6. One intriguing corollary of this view is that the de novoformation of centrioles is possible in most if not all cell types, unless it is actively suppressed by pre-existing centrioles. In this context, it will be interesting to clarify the role of pericentriolar material (PCM) components associated with those centrioles in the spatial and numerical control of centriole assembly11–13.

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947860/

1) http://rstb.royalsocietypublishing.org/content/369/1650/20130452

y

The central event of mitosis—chromosome segregation—depends in all eukaryotes on a complex and beautiful machine called the mitotic spindle. The spindle is a bipolar array of microtubules, which pulls sister chromatids apart in anaphase, thereby segregating the two sets of chromosomes to opposite ends of the cell, where they are packaged into daughter nuclei. In most somatic animal cells, each spindle pole is focused at a protein organelle called the centrosome . The centrosome is the primary microtubule-organizing center (MTOC) in animal cells. Microtubules emanate from the Centrosome in Animal Cells. The centrosome is located near the nucleus  from which microtubules are nucleated at their minus ends, so the plus ends point outward and continuously grow and shrink, probing the entire three-dimensional volume of the cell. Centrosomes control the orientation of chromosomes before the split. They create a spindle of microtubules that line the pairs up at the midplane, then pull them apart. Within the centrosomes are two motors called centrioles, oriented perpendicular to one another,

It is a highly dynamic organelle without a membrane. It plays a key role in organizing the microtubule network of the cell, most notably the mitotic spindle during cell division . It is   formed by cylinder-shaped centrioles surrounded by a microtubule-organizing matrix, is a hallmark of animal cells.  The centrosome is ( beside planarians and possibly other flatworms ) essential for the development of all animal species described so far. Fungi and plants lack centrosomes and therefore use other MTOC structures to organize their microtubules. The centrosome controls essential cellular processes such as cell division, migration and polarity by anchoring microtubule nucleating factors and cell cycle regulators. It also plays a crucial role during development  through its ability to control nucleus positioning and cell division orientation, and by nucleating primary cilia.   The PCM serves furthermore as a platform for protein complexes that regulate organelle trafficking, and protein degradation. 14 groups of  proteins are essential  for PCM assembly and function. Centrosome abnormalities are frequently seen in a variety of cancers, suggesting that dysfunction of this organelle may contribute to malignant transformation.


Centrioles are among the most beautiful of biological structures. How their highly conserved nine-fold symmetry is generated is a question that has intrigued cell biologists for decades. They are essential for both centrosome formation and cilium biogenesis in most eukaryotes.

The duplication of eukaryotic cells is an all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. Mitotic cell division is the most fundamental task of all living cells. Cells have intricate and tightly regulated machinery to ensure that mitosis occurs with appropriate frequency and high fidelity.

The only way to make a new cell is to duplicate a cell that already exists. A cell reproduces by performing an orderly sequence of events in which it duplicates its contents and then divides in two.  This cycle of duplication and division, known as the cell cycle, is the essential mechanism by which all living things reproduce. Dividing cells must coordinate their growth. a complex network of regulatory proteins trigger the different events of the cycle.  During the cell cycle, 18 different regulators are required, which order and coordinate the process. Each of these regulators are absolutely essential. If one is missing, the cell cycle is not completed and, the cell cannot duplicate.  Any of these regulators have only use if fully integrated into the process. They have no use or function by themselves. 

Centrosomes play a key role in organizing the microtubule network of the cell, most notably the mitotic spindle during cell division. 

The choreography of microtubules, centrosomes, and chromosomes during mitosis and meiosis is beautifully designed and uses finely regulated and synchronized movements. 

The centrosome is a structure, consisting of a pair of cylindrical microtubule-based organelles called centrioles, embedded in an amorphous network of proteins known collectively as Pericentriolar Material (PCM). Microtubules (MTs) originate from the PCM.  The PCM comprises a porous structural scaffold onto which γ-tubulin and other soluble components from the cytoplasm are loaded. Centrosome growth is an aggregation process of a condensed phase of PCM components, which segregate from the cytosol. The aggregation process leads to a centrosome phase that coexists with the cytosol and does rearrange internally. This implies that the centrosome phase is viscoelastic, such that on long timescales it behaves as a liquid-like material.

Cep192 is a pericentriolar protein that accumulates at centrosomes during mitosis and is required for PCM recruitment, centriole duplication, microtubule nucleation, and centrosome maturation.

Centrioles are among the most beautiful of biological structures. How their highly conserved nine-fold symmetry is generated is a question that has intrigued cell biologists for decades. 
Centrioles are present in all eukaryotic species that form cilia and flagella but are absent from higher plants and higher fungi which do not have cilia.
It seems likely that they have the primary purpose of growing cilia and flagella, which are important sensory and motile organelles found in almost all cells of the human body.  These organelles have many important functions in cells, and their dysfunction has been linked to a plethora of human pathologies, ranging from cancer to microcephaly to obesity.  Great progress has been made recently in understanding how these proteins interact and how these interactions are regulated to ensure that a new centriole is only formed at the right place and at the right time.

Centriole biogenesis requires 13 essential molecules. If any of these molecules is missing, centrioles cannot be made. Centriole assembly is also tightly regulated and abnormalities in this process can lead to developmental defects and cancer. Initiation of centriole duplication is under tight regulation to ensure the control of centriole number. Presumably, in centriole initiation, there is some form of cooperativity or positive feedback that results in asymmetric accumulation of the relevant proteins in a symmetric background. 

So we have not only the requirement of 18 regulators required for cell cycle regulation but also 13 essential molecules for centriole biogenesis, which by itself is also tighthly regulated, requiring positive feedback. 

It appears at the initial stage of the centriole assembly process as the first ninefold symmetrical structure. The cartwheel was first described more than 50 years ago, but it is only recently that its pivotal role in establishing the ninefold symmetry of the centriole was demonstrated.  This is a highly ordered structure that really stands out from the background. Constructed of rod-like microtubules, most centrioles have a nine-fold pattern, nine triplets or doublets evenly spaced at the rim, giving it a "cartwheel" appearance in cross-section. The comparison to a human-made cartwheel is evident, and so that it is intelligently designed. 

Obviously, the question arises, how could all this emerge gradually?  

Another amazing fact is that Electromagnetics play an important role in cell functioning and especially in cell duplication and division (mitosis). 

Recent development in the field of quantum biology highlights that the intracellular electromagnetic field (EMF) of microtubules plays an important role in many fundamental cellular processes such as mitosis. It is an intriguing hypothesis that centrosome functions as molecular dynamo to generate electric flow over the microtubules, leading to the electric excitation of microtubule EMF that is required for spindle body microtubule self-assembly. With the help of motors proteins within the centrosome, centrosome transforms the energy from ATP into intracellular EMF in the living cell that shapes the functions of microtubules. There will be a general impact for the cell biology field to understand the mechanistic function of centrosome for the first time in correlation with its structural features. 

The electromagnetic property of microtubule has been reported with both computation modelling and experimental evidences. 

To transform the chemical energy in ATP into electric magnetic field within the living cell, cell needs to have a molecular dynamo to transform the mechanistic movement of protein complexes to directional movements of intracellular electrons, leading to the electric excitation of the spindle body microtubules as well as the M phase chromosomes, which is essential for mitosis

We hypothesize that the centrosome functions as a molecular dynamo in the living cell to generate electric current from the cytosol electrolyte to the spindle body and M phase chromosome, leads to the electric excitation of the spindle body and chromosome during mitosis.

Taken together the longitudinal, or axial, vibration of the 13 filaments of an MT and then the 27 MTs making up the centriole barrel produce the electromagnetic field surrounding the centriole . Interestingly, this field is also found to be ferromagnetic. Also of interest, the fundamental vibration frequency of an MT filament is approximately 465 MHz, although this frequency is continually changing due to the ongoing length changing of the filaments. The electropolarity of the centrioles enables them to exert forces at a distance—that is, forces without physical contact.

All this indicates the requirement of forsight to produce all these ingenius mechanisms,and intelligence.