The astonishing language written on microtubules, amazing evidence of design

The astonishing  language is written on microtubules, amazing evidence of  design

https://reasonandscience.catsboard.com/t2096-the-astonishing-language-written-on-microtubules-amazing-evidence-of-design

The following information is truly mind-boggling. Take your time to read all through, and check the links. The creator of life has left a wealth of evidence of his existence in creation. A treasure grove to evidence intelligent design is every living cell. Its widely known that DNA is an advanced information storage device, encoding complex specified information to make proteins and directing many highly complex processes in the cell. What is less known, is that there are several other code systems as well, namely the histone binding code, transcription factor binding code, the splicing code, and the RNA secondary structure code. And there is another astonishing code system, called the tubulin code, which is being unraveled in recent scientific research. It is known so far that amongst other things, it directs and signals Kinesin and Myosin motor proteins precisely where and when to disengage from nanomolecular superhighways and deliver their cargo.

https://reasonandscience.catsboard.com/t1448-kinesin-motor-proteins-amazing-cargo-carriers-in-the-cell?highlight=kinesin

Recent research holds that this code in an amazing manner even stores our memories in the brain and makes them available in the long term.

https://reasonandscience.catsboard.com/t2182-heres-an-incredible-idea-for-how-memory-works#4032

For cells to function properly, they must organize themselves and interact mechanically with each other and with their environment. They have to be correctly shaped, physically robust, and properly structured internally. Many have to change their shape and move from place to place. All cells have to be able to rearrange their internal components as they grow, divide, and adapt to changing circumstances. These spatial and mechanical functions depend on a remarkable system of filaments called the cytoskeleton. The cytoskeleton’s varied functions depend on the behavior of three families of protein filaments—actin filaments, microtubules, and intermediate filaments. Microtubules are very important in a number of cellular processes. They are involved in maintaining the structure of the cell and provide a platform for intracellular macromolecular assemblies through dynein and kinesin motors. They are also involved in chromosome separation (mitosis and meiosis) and are the major constituents of mitotic spindles, which are used to pull apart eukaryotic chromosomes. Mitotic cell division is the most fundamental task of all living cells. Cells have the intricate and tightly regulated machinery to ensure that mitosis occurs with appropriate frequency and high fidelity. If someone wants to explain the origin of eukaryotic cells, the arise of mitosis and its mechanism and involved cell organelles and proteins must be elucidated. The centrosome plays a crucial role: it functions as the major microtubule-organizing center and plays a vital role in guiding chromosome segregation during mitosis. In the centrosome, two centrioles reside at right angles to each other, connected at one end by fibers.
These architecturally perfect structures are essential in many animal cells and plants (though not in flowering plants or fungi, or in prokaryotes). They help organize the centrosomes, whose spindles of microtubules during cell division reach out to the lined-up chromosomes and pull them into the daughter cells.

https://reasonandscience.catsboard.com/t2090-centriole-centrosome-the-centriole-spindle-the-most-complex-machine-known-in-nature?highlight=spindle

α- and β-tubulin heterodimers are the structural subunits of microtubules. The structure is divided in the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins. Unless all 3 functional domains were fully functional right from the beginning,  tubulins would have no useful function. There would be no reason for the Taxol-binding site to be without motor proteins existing. Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function.

https://reasonandscience.catsboard.com/t2096-the-cytoskeleton-microtubules-and-post-translational-modification#4033

Microtubule dynamics inside the cell are governed by a variety of proteins that bind tubulin dimers or microtubules. Proteins that bind to microtubules are collectively called microtubule-associated proteins, or MAPs.The MAP family includes large proteins like MAP-1A, MAP-1B, MAP-1C, MAP-2, and MAP-4 and smaller components like tau and MAP-2C.

This is highly relevant. Microtubules depend on microtubule-associated proteins for proper function. Interdependence is a hallmark of intelligent design and strong evidence that both, microtubules, and MAP's had to emerge together, at the same time, since one depends on the other for proper function. But more than that. Microtubules are essential to form the cytoskeleton, which is essential for cell shape and structure. In a few words, No MAP's, no proper function of microtubules. No microtubules, no proper function of the cytoskeleton. No cytoskeleton, no proper functioning cell. The evidence is very strong, that all these elements had to arise together at once. Kinesin and Dynein belong to MAP proteins. Kinesin-13 proteins contribute to microtubule depolymerizing activity to the centrosome and centromere during mitosis. These activities have been shown to be essential for spindle morphogenesis and chromosome segregation.  A step-wise evolutionary emergence of eukaryotic cells is not feasible since several parts of the call can only work if interacting together in an interlocked fully developed system.

When incorporated into microtubules, tubulin accumulates a number of post-translational modifications, many of which are unique to these proteins. These modifications include detyrosination, acetylation, polyglutamylation, polyglycylation,phosphorylation, ubiquitination, sumoylation, and palmitoylation. The α- and β-tubulin heterodimer undergoes multiple post-translational modifications (PTMs). The modified tubulin subunits are non-uniformly distributed along microtubules. Analogous to the model of the ‘histone code’ on chromatin, diverse PTMs are proposed to form a biochemical ‘tubulin code’ that can be ‘read’ by factors that interact with microtubules.

This is a relevant and amazing fact and raises the question of how the " tubulin code " beside the several other codes in the cell emerged. In my view, once more this shows that intelligence was required to create these amazing biomolecular structures; the formation of coded information has always shown to be able only to be produced by intelligent minds. What good would the tubulin code be for, if no specific goal was foreseen, that is, it acts as an emitter of information, and if there is no destination and receiver of the information, there is no reason for the code to arise in the first place? So both, sender and receiver, must exist first as hardware, that is the microtubules with the post-transcriptional modified tubulin units in a specified coded conformation, and then the receiver, which can be MAP's in general, or Kinesin or Myosin motor proteins, which are directed to the right destination to fulfill specific tasks, or other proteins directed for specific jobs.

Taken together, multiple and complex tubulin PTMs provide a myriad of combinatorial possibilities to specifically ‘tag’ microtubule subpopulations in cells, thus destining them for precise functions. How this tubulin or microtubule code allows cells to divide, migrate, communicate, and differentiate in an ordered manner is an exciting question that needs to be answered in the near future. Initial insights have already revealed the potential roles of tubulin PTMs in a number of human pathologies, like cancer, neurodegeneration, and ciliopathies. This raises the question: If PTM's are not precise and fully functioning, they cause diseases. What about if the MAP's are not fully specified and evolved? There is a threshold, a dividing line between a non-functional protein - amino acid sequence that is non-functional, and when it has enough residues to fold properly and become functional. How proteins arose in the first place is a mystery for the proponents of natural mechanisms..... Not only does it have to be elucidated how this tubulin or microtubule code allows cells to do all these tasks, but also what explains best it's arising and encoding. Most of these enzymes are specific to tubulin and microtubule post-translational modifications. They have only use if microtubules exist. Microtubules, however, require these enzymes to modify their structures.  It can, therefore, be concluded that they are interdependent and could not arise independently by natural evolutionary mechanisms. 

An emerging hypothesis is that tubulin modifications specify a code that dictates biological outcomes through changes in higher-order microtubule structure and/or by recruiting and interacting with effector proteins. This hypothesis is analogous to the histone code hypothesis ‑ that modifications on core histones, acting in a combinatorial or sequential fashion, specify multiple functions of chromatin such as changes in higher-order chromatin structure or selective activation of transcription. The apparent parallels between these two types of structural frameworks, chromatin in the nucleus and microtubules in the cytoplasm, are intriguing 

Isn't that striking evidence of a  common designer that invented both codes? 

https://reasonandscience.catsboard.com/t2096-the-cytoskeleton-microtubules-and-post-translational-modification#4035

Microtubules are typically nucleated and organized by dedicated organelles called microtubule-organizing centres (MTOCs). Contained within the MTOC is another type of tubulin, γ-tubulin, which is distinct from the α- and β-subunits of the microtubules themselves. The γ-tubulin combines with several other associated proteins to form a lock washer-like structure known as the γ-tubulin ring complex" (γ-TuRC). This complex acts as a template for α/β-tubulin dimers to begin polymerization; it acts as a cap of the (−) end while microtubule growth continues away from the MTOC in the (+) direction. The γ-tubulin small complex (γTuSC) is the conserved, essential core of the microtubule nucleating machinery, and it is found in nearly all eukaryotes.

This γ-tubulin ring complex is a striking example of a purposeful design that is required to nucleate the microtubules into the right shape. There would be no function for the γ-tubulin ring complex to emerge without microtubules since it would have no function on its own. Furthermore, it is made of several subunits that are indispensable for proper use, that is, for example, the attachment factors, accessory proteins, and γ-tubulins, which constitute an irreducible γ-tubulins ring complex, made of several interlocked parts, which could not emerge by natural selection. The complex has only a purposeful function when microtubules have to be assembled. So the γ-tubulins ring complex and microtubules are interdependent.

See its striking structure here :

https://reasonandscience.catsboard.com/t2096-the-cytoskeleton-microtubules-and-post-translational-modification#4040

Here’s an Incredible Idea For How Memory Works

Cytoskeletal Signaling: Is Memory Encoded in Microtubule Lattices by CaMKII Phosphorylation?

how the brain could store information long-term has been something of a mystery. But now researchers have developed a very interesting idea of how the brain’s neurons could store information using, believe it or not, a binary encoding scheme based on phosphorylation:

Memory is attributed to strengthened synaptic connections among particular brain neurons, yet synaptic membrane components are transient, whereas memories can endure. This suggests synaptic information is encoded and ‘hard-wired’ elsewhere, e.g. at molecular levels within the post-synaptic neuron. In long-term potentiation (LTP), a cellular and molecular model for memory, post-synaptic calcium ion (Ca2+) flux activates the hexagonal Ca2+-calmodulin dependent kinase II (CaMKII), a dodacameric holoenzyme containing 2 hexagonal sets of 6 kinase domains.
This enzyme has a astonishing and remarkable configuration and functionality :

Each kinase domain can either phosphorylate substrate proteins, or not (i.e. encoding one bit). Thus each set of extended CaMKII kinases can potentially encode synaptic Ca2+ information via phosphorylation as ordered arrays of binary ‘bits’. Candidate sites for CaMKII phosphorylation-encoded molecular memory include microtubules (MTs), cylindrical organelles whose surfaces represent a regular lattice with a pattern of hexagonal polymers of the protein tubulin. Using molecular mechanics modeling and electrostatic profiling, we find that spatial dimensions and geometry of the extended CaMKII kinase domains precisely match those of MT hexagonal lattices. This suggests sets of six CaMKII kinase domains phosphorylate hexagonal MT lattice neighborhoods collectively, e.g. conveying synaptic information as ordered arrays of six “bits”, and thus “bytes”, with 64 to 5,281 possible bit states per CaMKII-MT byte. Signaling and encoding in MTs and other cytoskeletal structures offer rapid, robust solid-state information processing which may reflect a general code for MT-based memory and information processing within neurons and other eukaryotic cells.

Size and geometry of the activated hexagonal CaMKII holoenzyme and the two types of hexagonal lattices (A and B) in MTs are identical. 6 extended kinases can interface collectively with 6 tubulins

Is the precise interface matching striking coincidence, or purposeful design ? Either a intelligent , goal oriented creator made the correct size, where CaMKII would fit and match the hexagonal lattices, or that is the result of unguided, random, evolutionary processes. What explanation makes more sense ?  

The electrostatic pattern formed by a neighborhood of tubulin dimers on a microtubule ( MT )  surface  shows highly negative charged regions surrounded by a less pronounced positive background, dependent on the MT lattice type . These electrostatic fingerprints are complementary to those formed by the 6 CaMKII holoenzyme kinase domains making the two natural substrates for interaction. Alignment of the CaMKII holoenzyme with tubulin dimers in the A-lattice MT arrangement yields converging electric field lines indicating a mutually attractive interaction.

So additionally to the precise interface matching significant association of the CaMKII holoenzyme with the MT through electrostatic forces indicates cumulative evidence of design.

there are 26 possible encoding states for a single CaMKII-MT interaction resulting in the storage of 64 bits of information. This case, however, only accounts for either α- or β-tubulin phosphorylation, not both. In the second scenario each tubulin dimer is considered to have three possible states – no phosphorylation (0), β-tubulin phosphorylation (1), or α-tubulin phosphorylation (2) (see Figure 5 B). These are ternary states, or ‘trits’ (rather than bits). Six possible sites on the A-lattice yield 36 = 729 possible states. The third scenario considers the 9-tubulin B-lattice neighborhood with ternary states. As in the previous scenarios the central dimer is not considered available for phosphorylation. In this case, 6 tubulin dimers out of 8 may be phosphorylated in three possible ways. The total number of possible states for the B lattice neighborhood is thus 36–28−8(27) = 5281 unique states.

So thirdly we have here a advanced encoding mechanism of information, which adds to the precise interface and electrostatic force interactions, which adds further cumulative evidence of design.

https://reasonandscience.catsboard.com/t2181-cell-communication-and-signalling-evidence-of-design#4019

Motor proteins dynein and kinesin move along microtubules (using ATP as fuel) to transport and deliver components and precursors to specific synaptic locations. While microtubules are assumed to function as passive guides, like railroad tracks for motor proteins, the guidance mechanism seems to be through CaMKII kinase enzymes which "write" on microtubules through phosphorylation and encode the way  to regulate motor protein transport along microtubules directly, and signal motor proteins precisely where and when to disengage from microtubules and deliver their cargo. There needs to be programming all the way along. Programming to make the specific enzymes, and how they have to operate.  That constitutes in my view another amazing argument for intelligent design. 




The Cytoskeleton,  microtubules, and post-translational modification

The Cytoplasm Is Organized by the Cytoskeleton and Is Highly Dynamic

Fluorescence microscopy reveals several types of protein filaments crisscrossing the eukaryotic cell, forming an interlocking three-dimensional meshwork, the cytoskeleton. There are three general types of cytoplasmic filaments—actin filaments, microtubules, and intermediate filaments.



differing in width (from about 6 to 22 nm), composition, and specific function. All types provide structure and organization to the cytoplasm and shape to the cell. Actin filaments and microtubules also help to produce the motion of organelles or of the whole cell. Each type of cytoskeletal component is composed of simple protein subunits that associate noncovalently to form filaments of uniform thickness. These filaments are not permanent structures; they undergo constant disassembly into their protein subunits and reassembly into filaments. Their locations in cells are not rigidly fixed but may change dramatically with mitosis, cytokinesis, amoeboid motion, or changes in cell shape. The assembly, disassembly, and location of all types of filaments are regulated by other proteins, which serve to link or bundle the filaments or to move cytoplasmic organelles along the filaments. The picture that emerges from this brief survey of eukaryotic cell structure is of a cell with a meshwork of structural fibers and a complex system of membraneenclosed compartments. The filaments disassemble and then reassemble elsewhere. Membranous vesicles bud from one organelle and fuse with another. Organelles move through the cytoplasm along protein filaments, their motion powered by energy-dependent motor proteins. The endomembrane system segregates specific metabolic processes and provides surfaces on which certain enzyme-catalyzed reactions occur. Exocytosis and endocytosis, mechanisms of transport (out of and into cells, respectively) that involve
membrane fusion and fission, provide paths between the cytoplasm and surrounding medium, allowing for secretion of substances produced in the cell and uptake of extracellular materials.



Although complex, this organization of the cytoplasm is far from random. The motion and positioning of organelles and cytoskeletal elements are under tight regulation, and at certain stages in its life, a eukaryotic cell undergoes dramatic, finely orchestrated reorganizations, such as the events of mitosis. The interactions between the cytoskeleton and organelles are noncovalent, reversible, and subject to regulation in response to various intracellular and extracellular signals.

The Cytoskeleton

For cells to function properly, they must organize themselves in space and interact mechanically with each other and with their environment. They have to be correctly shaped, physically robust, and properly structured internally. Many have to change their shape and move from place to place. All cells have to be able to rearrange their internal components as they grow, divide, and adapt to changing circumstances. These spatial and mechanical functions depend on a remarkable system of filaments called the cytoskeleton. The cytoskeleton’s varied functions depend on the behavior of three families of protein filaments—actin filaments, microtubules, and intermediate filaments. Each type of filament has distinct mechanical properties, dynamics, and biological roles, but all share certain fundamental features. Just as we require our ligaments, bones, and muscles to work together, so all three cytoskeletal filament systems must normally function collectively to give a cell its strength, its shape, and its ability to move. In this chapter, we describe the function and conservation of the three main filament systems. We explain the basic principles underlying filament assembly and disassembly, and how other proteins interact with the filaments to alter their dynamics, enabling the cell to establish and maintain internal order, to shape and remodel its surface, and to move organelles in a directed manner from one place to another. Finally, we discuss how the integration and regulation of the cytoskeleton allows a cell to move to new locations.

FUNCTION AND ORIGIN OF THE CYTOSKELETON

The three major cytoskeletal filaments are responsible for different aspects of the cell’s spatial organization and mechanical properties. Actin filaments determine the shape of the cell’s surface and are necessary for whole-cell locomotion; they also drive the pinching of one cell into two. Microtubules determine the positions of membrane-enclosed organelles, direct intracellular transport, and form the mitotic spindle that segregates chromosomes during cell division. Intermediate filaments provide mechanical strength. All of these cytoskeletal filaments interact with hundreds of accessory proteins that regulate and link the filaments to other cell components, as well as to each other. The accessory proteins are essential for the controlled assembly of the cytoskeletal filaments in particular locations, and they include the motor proteins, remarkable molecular machines that convert the energy of ATP hydrolysis into mechanical force that can either move organelles along the filaments or move the filaments themselves. In this section, we discuss the general features of the proteins that make up the filaments of the cytoskeleton. We focus on their ability to form intrinsically polarized and self-organized structures that are highly dynamic, allowing the cell to rapidly modify cytoskeletal structure and function under different conditions.

https://sci-hub.tw/https://www.annualreviews.org/doi/full/10.1146/annurev-cellbio-100818-125149

Microtubules

Microtubules are very important in a number of cellular processes. They are involved in maintaining the structure of the cell and, together with microfilaments and intermediate filaments, they form thecytoskeleton. They provide platforms for intracellular transport and are involved in a variety of cellular processes, including the movement of secretory vesicles, organelles, and intracellular macromolecular assemblies ( dynein and kinesin).They are also involved in chromosome separation (mitosis and meiosis), and are the major constituents of mitotic spindles, which are used to pull apart eukaryotic chromosomes.


The ab tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes. 8 . The a- and b-tubulins share 40% amino-acid sequence identity, both exist in several isotype forms, and both undergo a variety of posttranslational modifications. The monomer structure is very compact, but can be divided into three functional domains: the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins.

Unless all 3 functional domais were fully functional right from the start, the tubulins would have no useful function. There would be no reason for the Taxol-binding site to be without motor proteins existing.





Of the structural elements found in the cytoskeleton, microtubules are the largest. A well-known microtubule-based cellular structure is the axoneme of cilia and flagella, the appendages responsible for motility of eukaryotic cells. We have already encountered an example of such a structure—the axoneme of the sperm tail shown in Figure 4-12 consists of microtubules. Microtubules also form the mitotic spindle fibers that separate chromosomes prior to cell division. Besides their involvement in motility and chromosome
movement, microtubules also play an important role in the organization of the cytoplasm and the intracellular movement of macromolecules and other materials in the cell. They contribute to the overall shape of the cell, the spatial disposition of its organelles, and the distribution of microfilaments and intermediate filaments. Examples of the diverse phenomena governed by microtubules include the asymmetric shapes of animal cells, the plane of cell division in plant cells, the ordering of filaments during muscle development, and the positioning of mitochondria around the axoneme of motile appendages.  Although microtubules are usually drawn as if they are straight and rigid, video microscopy reveals them to be quite flexible in living cells. The wall of the microtubule consists of longitudinal arrays of protofilaments, usually 13 of them arranged side by side around the hollow center, called the lumen. Each protofilament is a linear polymer of tubulin. Tubulin is a dimeric protein consisting of two similar but distinct polypeptide subunits, a-tubulin and b-tubulin. All of the tubulin dimers in each of the protofilaments are oriented in the same direction, such that all of the subunits face the same end of the microtubule. This uniform orientation gives the microtubule an inherent polarity. The polarity of microtubules has important implications for their assembly and for the directional movement of membrane-bounded organelles that microtubules are associated with.



Microtubules are structurally more complex than actin filaments, but they are also highly dynamic and play comparably diverse and important roles in the cell. Microtubules are polymers of the protein tubulin. The tubulin subunit is itself a heterodimer formed from two closely related globular proteins called α-tubulin and β-tubulin, each comprising 445–450 amino acids, which are tightly bound together by noncovalent bonds (Figure A below).



These two tubulin proteins are found only in this heterodimer, and each α or β monomer has a binding site for one molecule of GTP. The GTP that is bound to α-tubulin is physically trapped at the dimer interface and is never hydrolyzed or exchanged; it can therefore be considered to be an integral part of the tubulin heterodimer structure. The nucleotide
on the β-tubulin, in contrast, may be in either the GTP or the GDP form and is exchangeable within the soluble (unpolymerized) tubulin dimer. Tubulin is found in all eukaryotic cells, and it exists in multiple isoforms. Yeast and human tubulins are 75% identical in amino acid sequence. In mammals, there are at least six forms of α-tubulin and a similar number of β-tubulins, each encoded by a different gene. The different forms of tubulin are very similar, and they generally copolymerize into mixed microtubules in the test tube. However, they can have distinct locations in cells and tissues and perform subtly different functions. As a striking example, mutations in a particular human β-tubulin gene
give rise to a paralytic eye-movement disorder due to loss of ocular nerve function. Numerous human neurological diseases have been linked to specific mutations in different tubulin genes.

Microtubules Are Hollow Tubes Made of Protofilaments

A microtubule is a hollow cylindrical structure built from 13 parallel protofilaments, each composed of αβ-tubulin heterodimers stacked head to tail and then folded into a tube (Figure B–D above). Microtubule assembly generates two new types of protein–protein contacts. Along the longitudinal axis of the microtubule, the “top” of one β-tubulin molecule forms an interface with the “bottom” of the α-tubulin molecule in the adjacent heterodimer. This interface is very similar to the interface holding the α and β monomers together in the dimer subunit, and the binding energy is high. Perpendicular to these interactions, neighboring protofilaments form lateral contacts. In this dimension, the main lateral contacts are between monomers of the same type (α–α and β–β). As longitudinal and lateral contacts are repeated during assembly, a slight stagger in lateral contacts gives rise to the helical microtubule lattice. Because multiple contacts within the lattice hold most of the subunits in a microtubule in place, the addition and loss of subunits occurs almost exclusively at the microtubule ends. These multiple contacts among subunits make microtubules stiff and difficult to bend. The persistence length of a microtubule is several millimeters, making microtubules the stiffest and straightest structural elements found in most animal cells. The subunits in each protofilament in a microtubule all point in the same direction, and the protofilaments themselves are aligned in parallel

Microtubules Undergo Dynamic Instability

Microtubule dynamics, like those of actin filaments, are profoundly influenced by the binding and hydrolysis of nucleotide—GTP in this case. GTP hydrolysis occurs only within the β-tubulin subunit of the tubulin dimer. It proceeds very slowly in free tubulin subunits but is accelerated when they are incorporated into microtubules. Following GTP hydrolysis, the free phosphate group is released and the GDP remains bound to β-tubulin within the microtubule lattice. Thus, as in the case of actin filaments, two different types of microtubule structures can exist, one with the “T form” of the nucleotide bound (GTP) and one with the “D form” bound (GDP). The energy of nucleotide hydrolysis is stored as elastic strain in the polymer lattice, making the free-energy change for dissociation of a subunit from the D-form polymer more negative than the free-energy change for dissociation of a subunit from the T-form polymer. In consequence, the ratio of koff/kon for GDP-tubulin (its critical concentration [Cc(D)]) is much higher than that of GTP-tubulin. Thus, under physiological conditions, GTP-tubulin tends to polymerize and GDP-tubulin to depolymerize. Whether the tubulin subunits at the very end of a microtubule are in the T or the D form depends on the relative rates of GTP hydrolysis and tubulin addition. If the rate of subunit addition is high—and thus the filament is growing rapidly—then it is likely that a new subunit will be added to the polymer before the nucleotide in the previously added subunit has been hydrolyzed. In this case, the tip of the polymer remains in the T form, forming a GTP cap. However, if the rate of subunit addition is low, hydrolysis may occur before the next subunit is added, and the tip of the filament will then be in the D form. If GTP-tubulin subunits assemble at the end of the microtubule at a rate similar to the rate of GTP hydrolysis, then hydrolysis will sometimes “catch up” with the rate of subunit addition and transform the end to a D form. This transformation is sudden and random, with a certain probability per unit time that depends on the concentration of free GTP-tubulin subunits. Suppose that the concentration of free tubulin is intermediate between the critical concentration for a T-form end and the critical concentration for a D-form end (that is, above the concentration necessary for T-form assembly, but below that for the D form). Now, any end that happens to be in the T form will grow, whereas any end that happens to be in the D form will shrink. On a single microtubule, an end might grow for a certain length of time in a T form, but then suddenly change to the D form and begin to shrink rapidly, even while the free subunit concentration is held constant. At some later time, it might then regain a T-form end and begin to grow again. This rapid interconversion between a growing and shrinking state, at a uniform free subunit concentration, is called dynamic instability (Figure A below )



The change from growth to shrinkage is called a catastrophe, while the change to growth is called a rescue. In a population of microtubules, at any instant some of the ends are in the T form and some are in the D form, with the ratio depending on the hydrolysis rate and the free subunit concentration. In vitro, the structural difference between a T-form end and a D-form end is dramatic. Tubulin subunits with GTP bound to the β-monomer produce straight protofilaments that make strong and regular lateral contacts with one another. But the hydrolysis of GTP to GDP is associated with a subtle conformational change in the protein, which makes the protofilaments curved (Figure B). On a rapidly growing microtubule, the GTP cap is thought to constrain the curvature of the protofilaments, and the ends appear straight. But when the terminal subunits have hydrolyzed their nucleotides, this constraint is removed, and the curved protofilaments spring apart. This cooperative release of the energy of hydrolysis stored in the microtubule lattice causes the curled protofilaments to peel off rapidly, and curved oligomers of GDP-containing tubulin are seen near the ends of depolymerizing microtubules (Figure C).

Microtubule-associated proteins

MAPs bind to the tubulin subunits that make up microtubules to regulate their stability. A large variety of MAPs have been identified in many different cell types, and they have been found to carry out a wide range of functions. These include both stabilizing and destabilizing microtubules, guiding microtubules towards specific cellular locations, cross-linking microtubules and mediating the interactions of microtubules with other proteins in the cell.^[1]
Within the cell, MAPs bind directly to the tubulin dimers of microtubules. This binding can occur with either polymerized or depolymerized tubulin, and in most cases leads to the stabilization of microtubule structure, further encouraging polymerization. Usually, it is the C-terminal domain of the MAP that interacts with tubulin, while the N-terminal domain can bind with cellular vesicles, intermediate filaments or other microtubules. MAP-microtubule binding is regulated through MAP phosphorylation. This is accomplished through the function of the microtubule-affinity-regulating-kinase (MARK) protein. Phosphorylation of the MAP by the MARK causes the MAP to detach from any bound microtubules.^[2] This detachment is usually associated with a destabilization of the microtubule causing it to fall apart. In this way the stabilization of microtubules by MAPs is regulated within the cell through phosphorylation.

Role of microtubule-associated proteins in the control of microtubule assembly 12

In eukaryotic cells, microtubules, actin, and intermediate filaments interact to form the cytoskeletal network involved in determination of cell architecture, intracellular transport, modulation of surface receptors, mitosis, cell motility, and differentiation. Cytoskeletal organization and dynamics depend on protein self-associations and interactions with regulatory elements such as microtubule-associated proteins (MAPs). The MAP family includes large proteins like MAP-1A, MAP-1B, MAP-1C, MAP-2, and MAP-4 and smaller components like tau and MAP-2C.

This is highly relevant, and a explicit admission that microtubules depend on microtubule-associated proteins for proper function. Interdependence is a hallmark of intelligent design, and strong evidence that both, microtubules, and MAP's had to emerge together, at the same time, since one tepends on the other for proper function. But more than that. Microtubules are essental to form the cytoskeleton, which is essential for cell shape and structure. In a few words, No MAP's, no proper function of microtubules. No microtubules, no proper function of the Cytoskeleton. No Cytoskeleton, no proper functioning cell. Evidence is very strong, that all these elements had to arise together at ones. A stepwise  evolutionary emergence of eukaryotic cells is not feasable for one more reason, described here.   


Microtubule-Binding Proteins Modulate Filament Dynamics and Organization

Microtubule polymerization dynamics are very different in cells than in solutions of pure tubulin. Microtubules in cells exhibit a much higher polymerization rate (typically 10–15 μm/min, relative to about 1.5 μm/min with purified tubulin at similar concentrations), a greater catastrophe frequency, and extended pauses in microtubule growth, a dynamic behavior rarely observed in pure tubulin solutions. These and other differences arise because microtubule dynamics inside the cell are governed by a variety of proteins that bind tubulin dimers or microtubules. Proteins that bind to microtubules are collectively called microtubule-associated proteins, or MAPs. Some MAPs can stabilize microtubules against disassembly. A subset of MAPs can also mediate the interaction of microtubules with other cell components. This subset is prominent in neurons, where stabilized microtubule bundles form the core of the axons and dendrites that extend from the cell body. These MAPs have at least one domain that binds to the microtubule surface and another that projects outward. The length of the projecting domain can determine how closely MAP-coated microtubules pack together, as demonstrated in cells engineered to overproduce different MAPs. Cells overexpressing MAP2, which has a long projecting domain, form bundles of stable microtubules that are kept widely spaced, while cells overexpressing tau, a MAP with a much shorter projecting domain, form bundles of more closely packed microtubules. MAPs are the targets of several protein kinases, and phosphorylation of a MAP can control both its activity and localization inside cells.




Question : Had these microtubule binding proteins not have to exist right from the beginning, otherwise microtubules could and would not be able to exercise its function properly? 

Microtubule Plus-End-Binding Proteins Modulate Microtubule Dynamics and Attachments

Cells contain numerous proteins that bind the ends of microtubules and thereby influence microtubule stability and dynamics. These proteins can influence the rate at which a microtubule switches from a growing to a shrinking state (the frequency of catastrophes) or from a shrinking to a growing state (the frequency of rescues). For example, members of a family of kinesin-related proteins known as catastrophe factors (or kinesin-13) bind to microtubule ends and appear to pry protofilaments apart, lowering the normal activation-energy barrier that prevents a microtubule from springing apart into the curved protofilaments that are characteristic of the shrinking state



Another protein, called Nezha or Patronin, protects microtubule minus ends from the effects of catastrophe factors. While very few microtubule minus-end-binding proteins have been characterized, a large subset of MAPs has been identified that are enriched at microtubule plus ends. A particularly ubiquitous example is XMAP215, which has close homologs in organisms that range from yeast to humans. XMAP215 binds free tubulin subunits and delivers them to the plus end, thereby promoting microtubule polymerization and simultaneously counteracting catastrophe factor activity (see Figure above). The phosphorylation of XMAP215 during mitosis inhibits its activity and shifts the balance of its competition with catastrophe factors. This shift results in a tenfold increase in the dynamic instability of microtubules during mitosis, a transition that is critical for the efficient construction of the mitotic spindle. In many cells, the minus ends of microtubules are stabilized by association with a capping protein or the centrosome, or else they serve as microtubule depolymerization sites. The plus ends, in contrast, efficiently explore and probe the entire cell space. Microtubule-associated proteins called plus-end tracking proteins (+TIPs) accumulate at these active ends and appear to rocket around the cell as passengers at the ends of rapidly growing microtubules, dissociating from the ends when the microtubules begin to shrink The kinesin-related catastrophe factors and XMAP215 mentioned above behave as +TIPs and act to modulate the growth and shrinkage of the microtubule end to which they are attached. Other +TIPs control microtubule positioning by helping to capture and stabilize the growing microtubule end at specific cellular targets, such as the cell cortex or the kinetochore of a mitotic chromosome. EB1 and its relatives, small dimeric proteins that are highly conserved in animals, plants, and fungi, are key players in this process. EB1 proteins do not actively move toward plus ends, but rather recognize a structural feature of the growing plus end. Several of the +TIPs depend on EB1 proteins for their plus-end accumulation and also interact with each other and with the microtubule lattice. By attaching to the plus end, these factors allow the cell to harness the energy of microtubule polymerization to generate pushing forces that can be used for positioning the spindle, chromosomes, or organelles.

Tubulin-Sequestering and Microtubule-Severing Proteins Destabilize Microtubules

As it does with actin monomers, the cell sequesters unpolymerized tubulin subunits to maintain a pool of active subunits at a level near the critical concentration. One molecule of the small protein stathmin (also called Op18) binds to two tubulin heterodimers and prevents their addition to the ends of microtubules



Stathmin thus decreases the effective concentration of tubulin subunits that are available for polymerization, and enhances the likelihood that a growing microtubule will switch to the shrinking state. Phosphorylation of stathmin inhibits its binding to tubulin, and signals that cause stathmin phosphorylation can increase the rate of microtubule elongation and suppress dynamic instability. Stathmin has been implicated in the regulation of both cell proliferation and cell death. Interestingly, mice lacking stathmin develop normally but are less fearful than wild-type mice, reflecting a role for stathmin in neurons of the amygdala, where it is normally expressed at high levels. Severing is another mechanism employed by the cell to destabilize microtubules. To sever a microtubule, thirteen longitudinal bonds must be broken, one for each protofilament. The protein katanin, named after the Japanese word for “sword,” accomplishes this demanding task

Post-translational modifications of microtubules

When incorporated into microtubules, tubulin accumulates a number of post-translational modifications, many of which are unique to these proteins. These modifications include detyrosination, acetylation, polyglutamylation, polyglycylation,phosphorylation, ubiquitination, sumoylation, and palmitoylation. 



Posttranslation modification of tubulin is a key regulatory mechanism for adapting subsets of microtubules to specialized functions in eukaryotic cells 1



The microtubule network emerges from the centrosome and the pericentrosomal matrix allows the binding of γ-tubulin ring complexes (γTuRCs), formed by γ-tubulin and capping proteins. In association with microtubule plus end-tracking proteins (+TIPS), the γTuRCs provide stable plus ends that allow microtubules to grow. One such +TIPS, end binding 1 (EB1), facilitates the incorporation of α/β-tubulin heterodimers into a sheet formed by the tubulin protofilaments on GDP interchange by GTP in the β-tubulin subunit. EB proteins and proteins containing the TOG domain (XMAP125, CLASP) help fold the sheet into a tubule. The GTP then hydrolyzes into GDP and facilitates the catastrophe events that depolymerize the microtubules. However, ‘GTP seeds’ may remain to serve as a rescue focus in pre-existing microtubules. 2

Microtubules – polymers of tubulin – perform essential functions, including regulation of cell shape, intracellular transport and cell motility. 3 How microtubules are adapted to perform multiple diverse functions is not well understood. Post-translational modifications of tubulin subunits diversify the outer and luminal surfaces of microtubules and provide a potential mechanism for their functional specialization. Recent identification of a number of tubulin-modifying and -demodifying enzymes has revealed key roles of tubulin modifications in the regulation of motors and factors that affect the organization and dynamics of microtubules. 






The α- and β-tubulin heterodimer – the building block of microtubules – undergoes multiple post-translational modifications (PTMs) (Table above). The modified tubulin subunits are non-uniformly distributed along microtubules. Analogous to the model of the ‘histone code’ on chromatin, diverse PTMs are proposed to form a biochemical ‘tubulin code’ that can be ‘read’ by factors that interact with microtubules (Verhey and Gaertig, 2007).

This is a relevant and amazing fact , and raises the question of how the " tubulin code "  beside the several other codes in the cell emerged. In my view, once more this shows that intelligence was involved in creating these amazing biomolecular structures , since the formation of  coded information has always shown to be able only to be produced by intelligent minds. Furthermore: What good would the tubulin code be for, if no specific goal was in mind, that is, it acts as emitter , and if there is no destination of the information, there is no reason of the code to exist in the first place. So both, sender and receiver, must exist first as hardware, that is the microtubule with the post transcriptional modified tubulin units in a specified coded conformation, and the the receiver, which can be Kinesin or Myosin motor proteins, which are directed to the right destination, or other proteins.

In this Commentary, we will discuss recent advances in understanding the mechanism and functions of tubulin PTMs. Recent data provide strong support to a model that tubulin PTMs regulate microtubule effectors.

Most PTMs appear on tubulin subunits after polymerization into microtubules. One exception is phosphorylation on serine residue S172 of β-tubulin by the Cdk1 kinase that occurs on the unpolymerized tubulin dimer and inhibits its incorporation into assembling microtubules (Fourest-Lieuvin et al., 2006). Tubulin is also phosphorylated inside microtubules (Faruki et al., 2000; Gard and Kirschner, 1985; Laurent et al., 2004; Ma and Sayeski, 2007; Matten et al., 1990; Wandosell et al., 1987), presumably on tyrosine (Y) or S residues that are different from S172 on the β-subunit, but the sites and significance of the post-assembly phosphorylation events are not known. Tubulin PTMs that occur on microtubules and have been well characterized include acetylation of lysine (K) residues, detyrosination, glycylation and glutamylation (see below).

Microtubules are the largest filamentous components of the eukaryotic cytoskeleton. In spite of their extraordinary level of structural conservation, microtubules fulfill a vast range of different functions in cells. How this functional diversity is achieved remains an open question 4; however, recent advances point towards post-translational modifications (PTMs) of tubulin as a potent mechanism to generate microtubule identities. As many microtubule functions have direct implications for development and homeostasis of organisms, understanding the molecular functions of tubulin PTMs could provide a more differentiated view on the role of microtubules in both normal and pathological aspects of organism development.

Microtubules are involved in a vast array of cellular functions, including cell shape, motility and division, intracellular signaling and transport, cell differentiation, and generation of specific organelles. Microtubule functions can be regulated by modulating the biophysical parameters of the microtubules themselves, as well as by their interactions with specific microtubule-associated proteins (MAPs). Strikingly little is known about how specific MAPs can bind selectively to subsets of microtubules inside cells. One possible regulatory mechanism is the spatially and temporally restricted creation of microtubule identities by generating patterns of tubulin PTMs that are commonly referred to as the ‘tubulin code’.

Microtubules are assembled from evolutionarily conserved dimers of α- and β-tubulin that can be subjected to a broad range of PTMs. Some of these PTMs are ubiquitous protein modifications, such as acetylation, phosphorylation or palmitoylation, while others are less common, and some appear to be unique to tubulin. Among these rare modifications are the removal of gene-encoded amino acids by detyrosination and the follow-up deglutamylation of α-tubulin, or the addition of amino acids by tyrosination, polyglutamylation, or polyglycylation (Figure below).



Molecular localization of tubulin posttranslational modifications.
Microtubules are assembled from α-tubulin–β-tubulin dimers, which form hollow tubes composed of 13 protofilaments. The globular parts of the tubulins form the microtubule walls and the luminal surface, while the carboxy-terminal tails decorate the outer surface of microtubules, where many MAPs and motors bind. PTMs are found on various regions of the tubulin dimer: the α-tubulin K40 acetylation site is present at the luminal surface of microtubules, while the K252 acetylation site of β-tubulin is present on the boundary between α- and β-tubulins. Polyamination at Q15 and phosphorylation at S172 are found in the globular, folded part of β-tubulin and might influence the assembly rates and stability of microtubules. Detyrosination/tyrosination, Δ2- and Δ3-tubulin modulate the carboxy-terminal tails of α-tubulins, and polyglutamylation and polyglycylation are found within the carboxy-terminal tails of both α- and β-tubulin. All modifications of the carboxy-terminal tails are likely to regulate interactions between microtubules and associated proteins.

Signals generated by tubulin PTMs can give rise to different levels of information complexity. Simple modifications, such as acetylation, or detyrosination/tyrosination, generate binary signals, while polyglutamylation, polyglycylation, or polyamination can generate more graded signals due to variations in side-chain lengths and modification of either α- or β-tubulin or both. Most importantly, tubulin PTMs can affect different functional roles of microtubules depending on the localization of these modification sites. Acetylation, for instance, is found at the luminal surface of the microtubules, while most other tubulin PTMs modify the carboxy-terminal tails of tubulin that are located at the outer surface of microtubules (Figure above). As an important interaction site for many MAPs, the carboxy-terminal tail is thought to be a hotspot for the selective regulation of microtubule–MAP interactions. In this Primer, we discuss the various tubulin PTMs and their effects on microtubule functions.

Acetylation

To date, the most-studied tubulin modification is acetylation of lysine 40 (K40) of α-tubulin. The peculiarity of this modification site is its position at the luminal surface of microtubules. This feature renders it unlikely to regulate the binding of MAPs and motors to the outer surface of microtubules, while it is more likely to influence the binding of luminal proteins (Figure above). The anti-K40-acetylation antibody stains discrete microtubule populations in interphase cells and reveals that ciliary, flagellar and neuronal microtubules are strongly acetylated. K40 acetylation is catalyzed by the acetyl transferases αTAT/Mec-17 5 and Atat-2 (Atat-2 has so far only been found in Caenorhabditis elegans), and deacetylation is performed by the deacetylases HDAC6 and SIRT2 ( Figure below).



Enzymes involved in tubulin post-translational modifications.
Schematic representation of the known enzymes responsible for tubulin PTMs. The forward reactions (modifications) take place on microtubules, while the reverse reactions (demodifications) mostly affect soluble tubulin. Note that some reactions are irreversible, and some enzymes have not yet been identified. αTAT, α-tubulin N-acetyltransferase; HDAC6, histone deacetylase 6; SIRT2, sirtuin 2; TTL, tubulin tyrosine ligase; TTLL, TTL-like; CCP, cytosolic carboxypeptidase; TG, transglutaminase; CDK1, cyclin-dependent kinase  The color code corresponds to the previous Figure above.

Little is known about the functions of K40 acetylation. Initial observations suggesting a regulation of kinesin motors by microtubule acetylation at K40 could not be confirmed by later studies. In C. elegans, K40 acetylation is required in touch receptor neurons, and a recent study has shown that the modification is part of a mechanism that orients cells during three-dimensional migration.

A second acetylation event has recently been found on K252 of β-tubulin (Figure 1); this acetylation may negatively regulate microtubule assembly. Acetylation of K252 is catalyzed by the Sun acetyltransferase (Figure 2), which, in contrast to αTAT (the α-tubulin K40 acetylase) modifies free tubulin dimers and negatively regulates their assembly into microtubules. The possibility that tubulins are subject to more complex acetylation events was evoked by the identification of a number of potential acetylation sites on both α- and β-tubulin in a whole-proteome mass spectrometry study. None of the sites identified in this study has so far been confirmed by cell biological or biochemical approaches, yet some of these sites are especially intriguing because they are localized at the boundaries between α- and β-tubulin subunits and could thus have an important impact on microtubule assembly.



Detyrosination/tyrosination

The enzymatic, ribosome- and tRNA-independent incorporation of tyrosine into α-tubulin was discovered in 1973 and shown to be reversible in 1977. Sequencing of the α-tubulin genes at the end of the 1970s revealed that tyrosine is actually encoded by the α-tubulin gene — thus, the initial modification is detyrosination. Amazingly, the enzyme catalyzing detyrosination has so far not been discovered, while the enzyme catalyzing the reverse reaction, tubulin tyrosine ligase (TTL; Figure above), was the first tubulin-modifying enzyme to be identified. Biochemical and structural work has demonstrated that TTL exclusively modifies unpolymerized tubulin, which implies that the detyrosination/retyrosination cycle of tubulin depends critically upon the dynamic instability of microtubules. This mechanism, together with the preference of the tubulin-detyrosinating enzyme for polymerized microtubules, results in an accumulation of detyrosinated tubulin in stable microtubules.

Removal of the carboxy-terminal tyrosine from the α-tubulin protein exposes the penultimate glutamate residue , which is the genesis of the original nickname for detyrosinated tubulin, ‘Glu-tubulin’. This nickname has led to some confusion, however, since the discovery of tubulin glutamylation; detyrosinated tubulin is now usually called detyr-tubulin. Tyrosinated tubulin is consistently called tyr-tubulin.

In most organisms studied to date, only one TTL enzyme is present in the genome, and removal of this enzyme in mice led to a huge increase in detyr-tubulin (and Δ2-tubulin;) in cells. Moreover, TTL-knockout mice die at the perinatal stage due to neuronal abnormalities, which most likely result from mislocalization of the microtubule plus-end tracking protein (+TIP) CLIP170. The function of detyrosination is linked to its ability to modulate microtubule–MAP interactions: detyrosination enhances kinesin-1-driven transport in neurons, while it inhibits the microtubule plus-end localization of a subgroup of +TIPs — the CAP-Gly domain proteins. Moreover, detyrosination negatively regulates the activity of MCAK, a microtubule-depolymerizing kinesin. Detyrosinated tubulin is present at high levels in neuronal microtubules and other long-lived microtubule populations, but a direct causal relationship between detyrosination and microtubule stability has not been established.

The tyrosination status of microtubules has many functional implications. It is important for processes that depend on +TIP functions and can thus influence spindle orientation or growth cone guidance in neuronal pathfinding. Tyrosination levels also regulate microtubule dynamics and turnover in cells. The impact of detyrosination on kinesin-1 traffic, though subtle, could have a huge impact on neuronal transport due to the great distances that need to be covered in axons.

A pathological side effect of the detyrosination/retyrosination cycle could be the incorporation of nitrotyrosine into tubulin. Indeed, in a number of human disorders, the presence of nitric oxide leads to the nitration of free cellular tyrosine, which can be incorporated into α-tubulin by TTL. In contrast to tyr-tubulin, nitrotyrosinated tubulin cannot be detyrosinated, thus impacting on the balance between tyr- and detyr-tubulin, and potentially on the functions of the affected microtubules.

Δ2- and Δ3- tubulin

Following detyrosination, the carboxy-terminal tail of α-tubulin can be further modified by the removal of the penultimate glutamate residue, generating Δ2-tubulin (Figure 1). This deglutamylation reaction is catalyzed by enzymes from the cytosolic carboxypeptidase (CCP) family, which are also involved in the removal of post-translationally added polyglutamylation (Figure 2; see below). Continued proteolysis of the carboxy-terminal tubulin tail generates Δ3-tubulin, which suggests that further carboxy-terminal degradation of tubulin tails might be possible (Figure 1). Δ2-tubulin cannot be retyrosinated, thus irreversibly locking tubulin in the non-tyrosinatable status, which could be one of the possible functions of this PTM. It is tempting to speculate that further degradation of tubulin tails could remove the carboxy-terminal modification sites for other PTMs, such as polyglutamylation and polyglycylation.

Polyglutamylation

Polyglutamylation is the enzymatic addition of side chains of glutamates onto gene-encoded glutamate residues of the modified proteins (Figure 1). In α- and β-tubulins, polyglutamylation occurs on several sites within the carboxy-terminal tails, and side chains of various lengths are generated. The identification of the glutamylating enzymes revealed that the final polyglutamylation patterns on microtubules are determined by the activities of enzymes involved in the modification. Polyglutamylation is catalyzed by members of the tubulin tyrosine ligase-like (TTLL) family, each of which has a particular reaction preference — α- vs. β-tubulin and initiation vs. elongation of the glutamate side chains. Polyglutamylation is a reversible modification, and enzymes removing glutamate side chains belong to the cytosolic carboxypeptidase (CCP) family. Similar to the glutamylases, some deglutamylases preferentially shorten long glutamate chains, whereas other enzymes can specifically remove the branching point of glutamate side chains (Figure above).

Microtubule polyglutamylation affects the charges on the carboxy-terminal tails of tubulins and is thus believed to regulate electrostatic microtubule–MAP interactions. The first regulatory mechanism to be identified for microtubule polyglutamylation is microtubule-severing catalyzed by the enzyme spastin, the activity of which is much greater on polyglutamylated than on non-modified microtubules. This mechanistic study indicates that the mass and length of microtubules in cells may be regulated by subtle changes in microtubule polyglutamylation; also, preferred microtubule-severing sites might be determined by modification marks. Other proteins likely to be regulated by glutamylation are molecular motors and MAPs that bind specifically to the carboxy-terminal tubulin tails, but no experimental support for this is thus far available.

Glutamylation can be found in many cell types and on many cellular microtubule subsets; however, modification levels vary markedly. Particularly high levels have been found on centrioles, on the axonemes of cilia and flagella, and in neurons. During cell division, glutamylation levels are temporarily increased on the central mitotic spindle and on the midbody. Hyperglutamylation has been linked to neurodegeneration in a mouse model for Purkinje cell degeneration (pcd), underscoring the importance of balanced levels of microtubule polyglutamylation for neurons. Moreover, polyglutamylation has been reported to regulate the beating of motile cilia in different model organisms. Depletion of selected polyglutamylases in the protists Chlamydomonas reinhardtii and Tetrahymena thermophilia and in mouse ependymal cells led to a severe perturbation of ciliary beating, although cilia did not disassemble. Thus, polyglutamylation, and even further, specific polyglutamylation enzymes, are required for proper regulation of flagellar dynein and thus ciliary beating. The particularly high polyglutamylation levels found on mammalian centrioles have also been suggested to stabilize centrosomes, especially when external forces are exerted during cell division.

Polyglycylation

Polyglycylation is a tubulin modification analogous to polyglutamylation: it creates side chains of glycine, potentially using the same acceptor glutamate residues on target proteins. Glycylation modifies both α- and β-tubulins, has several modification sites within the carboxy-terminal tubulin tails, and generates chains of different lengths (Figure 1). Glycylases are members of the TTLL family and, as for glutamylases, in mammals they can be subdivided into initiating and side-chain-elongating glycylases (Figure 2). However, in Drosophila the enzymes are multifunctional, performing both initiation and elongation.

To date, glycylation has been found exclusively in axonemes of cilia and flagella, and little is known about its function in mammals. Glycylation has been shown to be crucial for the stability and maintenance of axonemes in Drosophila melanogaster sperm tails, which, in the absence of the testis-specific glycylase enzyme, disassemble completely during the maturation process. Similarly, co-depletion of the two initiating glycylases in mouse ependymal cells leads to a complete disassembly of motile cilia. Strikingly, while glycine side chains are elongated to generate polyglycylation in most organisms, the essential functions of the poly-modification seem to be sufficiently fulfilled by short glycine side chains. Curiously, although present in other primates, the enzymatic ability to generate polyglycylation is absent in humans as a result of two specific amino acid changes within the human genome.

Polyamination

Tubulin polyamination is a recently described irreversible PTM involving the addition of amines to glutamine residues of tubulin (Figure 1). The enzyme responsible for tubulin polyamination is a transglutaminase that can polyaminate both free tubulin and microtubules (Figure 2). A biochemical peculiarity of this modification is the fact that it adds positive charges to the overall acidic tubulin.

The amination modification was overlooked until recently because, in the classical tubulin purification protocol, aminated tubulin is excluded. Polyaminated microtubules are highly stable in cold- or calcium-induced depolymerization conditions, and thus polyamination is believed to contribute to the stability of microtubule subpopulations in neurons. Consistently, polyamination sites are found either close to the GTP-binding pocket of β-tubulin, or at the α-tubulin–β-tubulin dimer boundary.

Phosphorylation

Early biochemical studies have shown that tubulin can be phosphorylated, although only one precise phosphorylation site has been functionally analyzed so far. Phosphorylation of the serine residue S172 of β-tubulin has been shown to influence microtubule dynamics (Figure 1). This phosphorylation is catalyzed by cyclin-dependent kinase 1 (CDK1; Figure 2), and thus has been suggested to regulate microtubule behavior during cell division. Another intriguing, but less fully characterized, phosphorylation event is catalyzed by the tyrosine kinase Syk, which phosphorylates an unidentified residue within the carboxy-terminal domain of α-tubulin. The position of the modification site is such that it may affect binding of MAPs to microtubules; however, no functional data have yet demonstrated this.

Other modifications

Several other PTMs have been identified on tubulin, but little functional insight has been gained, and follow-up studies have yet to be performed. For example, ubiquitination of tubulin has been implicated in the proteolytic degradation of misfolded tubulin in cells; and tubulin palmitoylation has been suggested to regulate microtubule–membrane interactions. Tubulin glycosylation, arginylation, methylation and sumoylation have also been reported, but no details of sites or relevant functions have been provided so far.

Perspectives

Taken together, multiple and complex tubulin PTMs provide a myriad of combinatorial possibilities to specifically ‘tag’ microtubule subpopulations in cells, thus destining them for precise functions. How this tubulin or microtubule code allows cells to divide, migrate, communicate and differentiate in an ordered manner is an exciting question that needs to be answered in the near future. Initial insights have already revealed the potential roles of tubulin PTMs in a number of human pathologies, like cancer, neurodegeneration and ciliopathies.

Not only does it have to be elucidated how this tubulin or microtubule code allows cells to divide, migrate, communicate and differentiate in an ordered manner, but also what explains best its arise. Most of these enzymes are specific to tubuline and microtubule post translational modifications. They have only use if microtubules exist. Microtubules however required these enzymes to modify their structures. It can therefore be concluded that they are interdependent and could not arise independently by natural evolutionary mechanisms.

12) http://www.ncbi.nlm.nih.gov/pubmed/7480164

Writing and Reading the Tubulin Code  7

Microtubules give rise to intracellular structures with diverse morphologies and dynamics that are crucial for cell division, motility and differentiation. They are decorated with abundant and chemically diverse posttranslational modifications that modulate their stability and interactions with cellular regulators. These modifications are important for the biogenesis and maintenance of complex microtubule arrays such as those found in spindles, cilia, neuronal processes and platelets. Here we discuss the nature and subcellular distribution of these posttranslational marks whose patterns have been proposed to constitute a tubulin code that is interpreted by cellular effectors. We review the enzymes responsible for writing the tubulin code, explore their functional consequences, and identify outstanding challenges in deciphering the tubulin code.

Microtubules are non-covalent cylindrical polymers formed by αβ-tubulin heterodimer building blocks. They possess two seemingly contradictory properties: they are highly dynamic, exhibiting rapid growth and shrinkage of their ends, but are also very rigid, with persistence lengths on the order of cellular dimensions. This duality is thought to underlie the versatile architectures of microtubule networks in cells and is tuned by a myriad of cellular effectors.

These fall into two categories: effectors that bind to the microtubule and alter its properties non-covalently (motors and microtubule associated proteins (MAPs)) and effectors that chemically modify the tubulin subunits (tubulin posttranslational modification enzymes). While the field has made tremendous progress in recent decades identifying a compendium of microtubule interacting proteins and understanding their interplay and regulation in the cell, we are just now starting to unravel the basic mechanisms used by cells to chemically modify microtubules, despite the fact that tubulin posttranslational modifications have been known for over forty years. A renaissance of interest into the roles of tubulin posttranslational modifications has been precipitated by the discovery in the last few years of the enzymes responsible for these modifications, methods for producing unmodified, engineered, as well as chemically defined modified tubulin, and developments and refinements of in vitro microtubule based assays using highresolution microscopy and microfabricated substrates. Tubulin posttranslational modifications are chemically diverse, ranging from phosphorylation, acetylation, palmitoylation, sumoylation,polyamination, and S-nitrosylation to tyrosination, glutamylation  and glycylation.

Most of these modifications are reversible. Tubulin posttranslational modifications are evolutionarily conserved and abundantly represented in cellular microtubules. Most importantly, their distribution is stereotyped in cells. For example, interphase microtubules are enriched in tyrosination, while kinetochore fibers and midbody microtubules are enriched in detyrosination and glutamylation. Axonal microtubules are enriched in detyrosination, acetylation and glutamylation, while the dynamic growth cone microtubules are enriched in tyrosination (Figs. 1A, B and C ). Microtubules in centrioles, cilia and flagella are especially heavily glutamylated . Glycylated microtubules are found predominantly in the axonemes of cilia and flagella , however some cytoplasmic microtubules in paramecia are also glycylated. The more morphologically complex microtubule arrays exhibit the largest diversity and abundance of tubulin posttranslational modifications like the microtubule arrays found in neurons, cilia or flagella or the highly specialized arrays found in some parasites such as toxoplasma and trypanosomes. In some cases, even adjacent microtubules have completely different posttranslational modification signatures. This is beautifully illustrated in axonemes where the B tubule is highly glutamylated, while the adjoining A tubule is not, but is enriched in tyrosination. Tubulin posttranslational modifications are also developmentally regulated. One striking example is during neuronal development that is accompanied by increases in glutamylation levels of both α- and β-tubulin, with β-tubulin glutamylation increasing mostly during the later stages of neuronal differentiation . The enzymes that introduce these conserved modifications are essential to normal development. Underscoring their importance for normal cell physiology increased levels of tubulin modifications are a hallmark of cancers and neurodegenerative disorders (reviewed in and several neurodevelopmental disorders are linked to mutations in tubulin genes at sites that could interfere with modification enzyme function. This microtubule chemical diversity was proposed to form the basis of a “tubulin code” that is read by cellular effectors. Despite the widespread appreciation for the ubiquity and functional importance of these modifications and their stereotyped distribution in organisms and cells, we do not currently understand how complex microtubule modification patterns are generated and what their functional consequences on cellular effectors are i.e. we do not understand how the tubulin code is written and interpreted by cells

Writers of the tubulin code: who are they?

The staggering chemical complexity of tubulin is produced by diverse protein families ranging from kinases and acetyltransferases to ATP-dependent ligases and carboxypeptidases. Many of these enzymes were not identified until the last decade. The first tubulin modification enzyme isolated and cloned was tubulin tyrosine ligase (TTL), the enzyme responsible for the ATPdependent re-addition of the genomically encoded tyrosine residue to the C-terminus of α-tubulin . TTL loss has drastic effects for the viability of the organism as TTL knockout mice die shortly after birth due to disorganized neuronal arrays. TTL suppression is also  strongly linked to tumorigenesis as well as tumor aggressiveness. The most abundant and variable components of the tubulin code, glutamylation and glycylation, are products of enzymes that belong to the tubulin tyrosine ligase-like (TTLL) family. Enzymes of this family share a core domain structurally homologous to TTL and an ATP-dependent amino acid ligation mechanism, which is also shared with more distantly related amino acid ligases such as glutathione-S-transferase or D-Ala:D-Ala ligase . All TTLLs preferentially modify microtubules, unlike TTL, which modifies soluble tubulin. Mammals encode thirteen TTLLs (Table 1). TTLL1, 4, 5, 6, 7, 9, 11 and 13 are glutamylases, while TTLL3, 8, and 10 are glycylases ; reviewed in. TTLL2 appears to be a glutamylase based on homology, but has yet to be biochemically characterized. TTLL12 is inactive both as a glutamylase and glycylase but is proposed to function as a pseudoenzyme that alters tubulin tyrosination and DNA methylation levels indirectly.


Writers of the tubulin code: specificity and combinatorial use

The complex microtubule modification patterns observed in cells are a function of the tissue distribution, developmental regulation and biochemical properties of tubulin posttranslational modification enzymes (i.e. substrate specificity and kinetic parameters) in addition to the tissue-specific enrichment of certain tubulin isoforms. In addition to these first order factors, pre-existing modifications and
their patterns may influence the further addition and removal of modifications. Furthermore, the preceding factors are convoluted with the dynamics of the microtubules themselves (that can potentially
be also influenced by modifications) and the effects of tubulin and microtubule binding proteins. Faced with this multilevel regulatory complexity, analysis requires the ability to generate chemically defined tubulin and microtubule substrates for in vitro reconstitution experiments. Such substrates can then be used to characterize the basic biochemical properties of the modifying enzymes. These defined substrates and enzymes will then allow quantitative investigation of the tubulin code ranging from the dynamics of the modified microtubules themselves to generation of temporal and
spatial microtubule modification patterns, to the effects on microtubule effectors. To date, the overwhelming majority of in vitro studies of microtubules and their regulators have employed tubulin purified from brain tissue. This tubulin is highly heterogeneous as it contains multiple posttranslational modifications (phosphorylation, acetylation, detyrosination, glutamylation) as well as multiple isoforms (eight α- and seven β-tubulin) that give rise to tens of different variants. While microtubules in cells show topographically defined modification patterns, the isolation procedure of microtubules from brain tissue results in complete scrambling of all the tubulin modifications and isoforms and thus makes the task of deciphering a tubulin code impossible. Recent advances now allow the purification of naïve, unmodified tubulin from various sources as well as recombinant tubulin in which posttranslational modification sites can be mutated. Using unmodified human tubulin, we have shown how to generate defined posttranslationally modified tubulin and microtubules that are tyrosinated, glutamylated and acetylated. Variable levels of glutamylation can be achieved and quantitatively measured using mass spectrometry.

How does the cell interpret the tubulin code?

Although the tubulin code is gradually yielding its secrets, what is not known is how the cell ultimately integrates the information encoded in tubulin posttranslational modifications. However, what is clear is that the organism invests a large amount of coding capacity for modification enzymes, that their loss can be deleterious to the organism and that nontrivial amounts of energy are expended to modify tubulin.  The power of the bottom-up reconstitution approach has been amply demonstrated in the last five decades in the study of basic cell biological processes. The analysis of tubulin posttranslational modifications is rapidly entering this stage. We see several major challenges: to characterize the dynamics and mechanical properties of modified microtubules, to understand the basic principles that give rise to the differential specificities of tubulin modification enzymes, to understand how motors and microtubule associated proteins are influenced by modifications and how their action in turn modulates the behavior of this dynamic polymer, to generate modification patterns that mimic those found in cells and build complex microtubule array geometries. These basic first steps should get us closer to understanding how the cell interprets the tubulin code.

A Tubulin Code

Microtubules can acquire a variety of evolutionarily conserved PTMs including polyglutamylation, polyglycylation, detyrosination (and related D2 modification), acetylation, phosphorylation and palmitoylation . In most cases, the modification enzymes act preferentially on tubulin subunits already incorporated into microtubules. One exception is the recently discovered phosphorylation of b-tubulin on Ser172 that occurs on unpolymerized tubulin in mitotic cells and inhibits incorporation of heterodimers into the polymer. An emerging hypothesis is that tubulin modifications specify a code that dictates biological outcomes through changes in higher-order microtubule structure and/or by recruiting and interacting with effector proteins. This hypothesis is analogous to the histone code hypothesis ‑ that modifications on core histones, acting in a combinatorial or sequential fashion, specify multiple functions of chromatin such as changes in higher-order chromatin structure or selective activation of transcription. The apparent parallels between these two types of structural frameworks, chromatin in the nucleus and microtubules in the cytoplasm, are intriguing and suggest that a general theme has evolved that regulates the functions of cellular polymers.



Isn't that  rather  striking evidence of a  common designer that invented both codes ?


One apparent parallel is that specific polymer regions can be distinguished biochemically and functionally by the presence of PTMs on their building blocks. Chromatin of genes active in transcription has increased acetylation on certain lysine residues of core histones. In a similar fashion, PTMs on tubulin are enriched in restricted subcellular areas and therefore have the potential to locally adapt microtubules for specific functions. For example, microtubules oriented towards a wound in a confluent monolayer of cells are enriched in detyrosination and acetylation and central spindle but not astral microtubules are marked by detyrosination, glutamylation and acetylation. A second parallel between chromatin and microtubules is that most PTMs take place on the tail of domains of histones and tubulins that comprise the outward face of the polymer (Fig. 1). In the case of a‑ and b-tubulin, most PTMs occur on the C-terminal tails (CTTs), essential domains that could not be resolved in atomic models but are known to comprise the binding region for a large number of microtubule binding proteins. In the paragraphs below, we will review recent work supporting the existence of a “tubulin code” and discuss potential ramifications.

What are the Enzymes that Establish the Tubulin Code?  9

The discovery of the enzymes that deposit the modifications has long lagged behind the discovery of the modifications themselves. However, the last few years have been a time of rapid progress in the identification of microtubule PTM enzymes. Detyrosination involves the enzymatic removal of the C-terminal tyrosine of a-tubulin by a carboxypeptidase. The identity of the tubulin carboxypeptidase has not been established despite multiple purification efforts. However, a recent study identified a novel cytosolic carboxypeptidase, Nna1/CCP1, that is abundant in tissues with high content of tubulin such as testis, pituitary and brain. Mice lacking Nna1/CCP1 lack detectable detyrosinated a-tubulin in mitral cells of the olfactory bulb and experience degeneration of Purkinje cells and altered gait which indicates that detyrosination could be important. Nna1/CCP1 belong to a family of six related genes with some showing restricted pattern of expression. Future biochemical studies should establish
whether Nna1/CCP1 is the long-sought tubulin carboxypeptidase. The enzyme that carries out the reverse reaction and converts soluble a-tubulin back to its unmodified form, tubulin tyrosine ligase (TTL), was identified much earlier. Interestingly, it appears that only mammals and trypanosomes have a TTL sequence in their genomes, while detyrosination is widespread among eukaryotes.

How are Patterns of Microtubule PTMs Established?

One major difference between the histone and tubulin codes may be in the way the information is propagated between generations of organelles. There is considerable evidence that the histone code can be inherited and maintained by copying the pattern from preexisting chromatin onto newly assembled chromatin at the time of DNA replication. The mechanism of this epigenetic transmission is likely based on the partitioning of preexisting histone particles to both strands of DNA during replication. Some microtubule-based
organelles (e.g., centrosomes and basal bodies) are inherited by a template-driven mechanism where new structures are formed in the vicinity of preexisting structures. However there is no evidence that the template organelle directly influences the PTM pattern in the newly formed organelle. Rather, the PTM pattern is recreated in the newly formed organelle in a gradual manner. For example, newly formed basal bodies and associated cilia have an immature pattern of PTMs characterized by shorter side chains of polyglycylation. Thus, the state of PTM distinguishes between old and new microtubule structures and could target assembly factors to forming organelles. Other microtubule-based structures, such as cytoplasmic microtubules, the mitotic spindle and cilia, are formed de novo mostly, if not entirely, from unmodified tubulin heterodimers. Thus, in case of both template-dependent and ‑independent microtubular structures, PTM patterns are probably recreated without a direct influence of preexisting PTMs. How then specific patterns of tubulin PTMs are established is unknown but three major regulatory mechanisms can be envisioned. One attractive mechanism of control involves spatial and temporal regulation of the activity of the PTM enzymes (both forward and reverse). For example, in wounded cell models, plasma membraneassociated members of the Rho, Rac and Cdc42 GTPase families trigger localized changes in both actin and microtubule dynamics that lead to cell polarization and directed motility. Importantly, downstream effectors of these GTPases include the microtubule plus-end tracking proteins (+TIPs) that “capture” and “stabilize” the ends of microtubules oriented towards the leading edge of the cell. Recent work has shown that activated versions of the +TIP proteins EB1, APC and CLASP can stimulate the formation of both detyrosinated and acetylated microtubules in wounded fibroblasts.
Yet whether GTPases and +TIP proteins directly impinge on the PTM enzymes has not been tested.

A second possible mechanism of regulation of PTM enzymes involves their subcellular localization. PGs1, a noncatalytic subunit of the TTLL1 a-tubulin glutamylase complex, localizes preferentially to major sites of tubulin glutamylation, notably centrosomes and basal bodies, axonemes, and the distal portion of neurites. Interestingly, this localization is regulated during the cell cycle as PGs1 localization is predominantly cytosolic during mitosis. In cultured neurons, the b-tubulin-preferring glutamylase TTLL7 is enriched in the somatodendritic regions and this localization correlates with the higher levels of glutamylation on b-tubulin in dendrites as compared to axons. The Ttll6Ap b-tubulin polyglutamylase specifically localizes to motile cilia in Tetrahymena and similar glutamylases localize to nonmotilesensory (primary)cilia in mammalian cells. Interestingly,
there are striking differences in the pattern of PTMs inside the cilium. For example, in doublet microtubules, detyrosination and both polymodifications occur mainly on the B-tubule while the A-tubule is largely unmodified. This could reflect the ability of modifying enzymes to associate with only a subset of microtubules and at specific positions within the lattice.
A third possibility is that the microtubule substrate is regulated in a way that controls their access or exposure time to PTM enzymes. In one scenario, the modification of subsets of microtubules is simply a time-dependent phenomenon, that is, microtubules that are “stabilized” remain in place long enough for the PTM enzymes to work. An alternative mechanism is that “stabilized” microtubules exist in an unknown structural state that makes them the preferred substrate for PTM addition. Indirect support for this possibility
comes from the fact that pharmacological treatments that stabilize microtubules (e.g., taxol) result in increased levels of several PTMs including detyrosination, acetylation and glycylation . It should also be considered that PTM patterns could be regulated by competition between PTM enzymes and other proteins that bind to similar sites on the microtubule polymer. Elucidation of the molecular mechanisms by which microtubule stability and the PTM enzymes are controlled, so far hindered by the lack of identification of the enzymes, will provide fertile ground for future work.

Who are the Interpreters of the Tubulin Code?

A major implication of the tubulin code is that PTMs influence the recruitment of protein complexes (microtubule effectors), which in turn contribute to microtubule-based functions. Three major classes of microtubule binding proteins can be considered as interpreters of the tubulin code. First, microtubule associated proteins (MAPs) such as Tau, MAP1 and MAP2 that bind statically along the length of microtubules. Second, plus end tracking proteins (+TIPs) that bind in a transient manner to the plus-ends of growing microtubules. And third, molecular motors that use the energy of ATP hydrolysis to carry cargoes along microtubule tracks. MAPs. Functional roles of structural MAPs are not completely understood but are thought to contribute to the stability and organization of microtubules, especially in neuronal cells. In vitro, Tau, MAP1B, and MAP2 bind preferentially to tubulins with moderate levels of polyglutamylation (~3 glutamyl units) whereas MAP1A shows optimal affinity for highly modified tubulins (~6 glutamyl units). As a-tubulin glutamylation is abundant in very young neurons whereas b-tubulin glutamylation increases during post-natal development, glutamylation could control transitions in MAP binding during neuronal development. Lys 40 a-tubulin acetylation may also influence MAP binding as overexpression of HDAC6 delocalized p58, a MAP involved in the association of Golgi membranes with microtubules.

+TIPs. Recent work has shown that tubulin detyrosination negatively affects the association of some +TIPs with microtubules. In yeast, removal of the C-terminal aromatic residue (phenylalanine) of a-tubulin disabled the interaction of Bik1p, a homolog of the mammalian cytoplasmic linker protein 170 (CLIP-170), with microtubule plus ends but had no effect on the association of Bim1p, the EB1 +TIP homolog. While it is not known whether such PTM occurs naturally in yeast, this experiment showed that the state of the C-terminal amino acid on a-tubulin has profound consequences in vivo. These results led to the hypothesis that the presence of unmodified a-tubulin at microtubule plus-ends plays an important role in localization of members of the CLIP-170 family of +TIP proteins. Indeed, in neurons and fibroblasts isolated from TTL-null mice, increased levels of detyrosination resulted in mislocalization of CLIP-170 and p150Glued whereas other +TIP proteins such as EB1 were unaffected.81,82 CLIP-170 and p150Glued both have a CAP-Gly domain. Structural work has shown that the CAP-Gly domain has a binding groove that may directly recognize the unmodified C-terminal sequence of a-tubulin. Taken together, these results indicate that +TIP proteins containing a CAP-Gly microtubule-binding domain require the presence of tyrosinated a-tubulin for their preferential localization to microtubule plus ends.

Motors. Studies in a wide variety of cell types have shown that cargoes delivered by motors can be targeted to specific subcellular destinations, such as cilia, axons or dendrites, and the leading edge of migrating fibroblasts. Furthermore, cargoes can even be targeted to subsets of microtubules within the mitotic spindle, the axon, and ciliary axoneme. Thus, the idea that microtubule PTMs could serve as “road signs” to direct motor transport to specific subcellular destinations has long been an attractive one.

How did the right road signs emerge ? Trial and error ? Up to what point does it make sense to believe that natural mechanisms produced the right information to direct cargo proteins ? What emerged first, the road signs, or the cargo proteins ? What good would be one without the other ?

Early studies showed that the addition of antibodies that specifically recognize detyrosinated tubulin prevented binding of Kinesin-1 to microtubules in vitro whereas antibodies to tyrosinated tubulin had no effect. Gel overlay and antibody inhibition experiments have shown that Kinesin-1 also binds preferentially to tubulin containing 3 glutamyl units. To directly examine the influence of PTMs on motors, recent experiments have utilized microtubules lacking specific PTMs due to genetic ablation of either the PTM sites or enzymes. Reed et al showed that loss of a-tubulin acetylation, a-tubulin detyrosination, or b-tubulin polymodifications resulted in decreased binding of Kinesin-1 to microtubules whereas loss of a-tubulin polymodifications had no effect. Acetylation of a-tubulin also positively regulates cytoplasmic dynein binding to microtubules. The effect of a-tubulin glutamylation has been examined using mice that lack functional PGs1, a noncatalytic subunit of TTLL1. The deficiency in a-tubulin glutamylation is associated with decreased binding of several motors to microtubules in vitro, however, the main effect in mutant (PGs1-/- ) brains and cells was on the subcellular distribution of the kinesin-3 motor Kif1A and its cargo synaptic vesicles. The possibility that decreased a-tubulin tyrosination in PGs1-/- mice could affect motor binding and motility, either directly or indirectly, cannot be ruled out presently. Further work is needed to elucidate the molecular mechanisms by which tubulin PTMs influence motor-microtubule interactions and motility. In particular, structural approaches are required to determine how the presence of PTMs affects the conformation of the polymer lattice.

What are the Biological Consequences of the Tubulin Code?

Intracellular trafficking. A role for tubulin modifications in directing intracellular trafficking was suggested early on based on microinjection of antibodies that recognize specific PTMs. Antibodies that specifically recognize detyrosinated tubulin inhibited two kinesin-dependent processes, the recycling of transferrin receptors to the plasma membrane and the extension of vimentin intermediate filaments. An antibody that recognizes mono‑ and polyglutamylated tubulin (GT335) interfered with kinesin-2-based pigment granule dispersion but not dynein-based aggregation in melanophores. With the identification of the enzymes that carry out tubulin modifications, more recent studies have used pharmacological or genetic methods to eliminate or enhance specific PTMs. Mice lacking functional TTL die soon after birth due to disorganization of neuronal networks and fibroblasts cultured from these mice show defects in cell morphology during interphase. Mice that are null for PGs1, a noncatalytic subunit of TTLL1 a-tubulin polyglutamylase, show mislocalization of synaptic vesicles, impaired synaptic transmission and disorganized axonemes of sperm flagella. In cultured neuronal cells, siRNA-mediated knockdown of TTLL7, a b-tubulin polyglutamylase, resulted in decreased neurite outgrowth. Surprisingly, elimination of acetylation in Chlamydomonas or Tetrahymena has no obvious phenotypic consequences and expression of a non-acetylatable a-tubulin in C. elegans rescues defects in neurons lacking MEC-12, the only identified tubulin in this organism that contains lysine at position 40.99-101 Thus, a-tubulin acetylation is not required for cell survival but recent work has demonstrated an important role for this PTM in differentiated cell types of vertebrates. Pharmacological inhibition of deacetylases results in hyperacetylation of microtubules that can affect a variety of intracellular trafficking events such as the selective transport of the Kinesin-1 cargo JIP1 to a subset of neurites, anterograde and retrograde transport of brain-derived neurotrophic factor (BDNF)-containing vesicles, dynein/dynactin transport of aggresomes,102,103 the exocytosis of interleukin (IL)-1b-containing secretory lysosomes, as well as cytoskeletal rearrangements at the immune synapse. Several studies have implicated a role for microtubule acetylation in cell motility—overexpression of HDAC6 leads to decreased acetylation and increased cell motility whereas inhibition of HDAC6 results in increased acetylation and decreased motility. One of the potential mechanisms by which HDAC6 contributes to cell motility was revealed in a recent report showing that HDAC6-inhibited migrating cells have decreased microtubule dynamics and decreased focal adhesion turnover. Taken together, these studies have provided important new advances in support of a tubulin code that directs intracellular trafficking

Assembly and motility of cilia. Polyglycylation is a conserved PTM that is abundant in cell types with cilia. In the ciliated protist Tetrahymena, polyglycylation appears to be essential based on experiments in which a‑ or b-tubulin genes were replaced by mutated versions that lack modification sites. While elimination of polyglycylation sites on a-tubulin had no effect, elimination of polyglycylation sites on the b-tubulin CTT was lethal. Strains with reduced levels of glycylation resulted in defects in axonemal structure, ciliary motility and cytokinesis, Strikingly, glycylation site-deficient mutants had specific defects in the axoneme, including defects in assembly of the central pair microtubules and in B-tubule assembly. These studies indicate that tubulin glycylation plays an important role in assembly of axonemal microtubules. One limitation to these studies is that ciliary tubulins are also extensively polyglutamylated on their CTTs. The respective roles of polyglutamylation and polyglycylation in the assembly of cilia need to be dissected and this task can now be attempted by direct manipulation of specific forward enzymes (TTLLs). Glutamylation and glycylation likely also play important roles in regulation of ciliary beating once the organelle is assembled as antibodies that recognize either polyglutamate or polyglycine side chains interfered with ciliary beating in ATP-reactivated axonemes.

Microtubule dynamics. There is no evidence that tubulin PTMs affect the intrinsic properties of microtubules such as their dynamicity. Yet several lines of evidence indicate that tubulin modifications may affect microtubule dynamics in vivo, possibly by regulating effectors that are important for turnover of microtubules. First, glutamylation may be important for the structural stability of centrioles. Second, some reports have indicated that inhibition of HDAC6 tubulin deacetylase led to increased microtubule  stability in vivo although other studies have failed to detect such effects. The differences between these studies could be related to the use of assays with different levels of sensitivity. Third, recent experiments have unraveled a relationship between microtubule PTMs and microtubule severing. Katanin and spastin are AAA type ATPases that regulate microtubule dynamics by severing microtubules. Mutations in spastin are responsible for the most frequent form of hereditary spastic paraplegia, a human neurodegenerative disease. Katanin and spastin require the CTT domains of tubulins for severing activity and spastin strongly interacts with the CTT of a-tubulin.Thus, it is possible that PTMs located on CTTs regulate the activity of severing factors. In support of this, mutation of a glutamate residue on the CTT of b-tubulin in C.elegans suppressed the lethal phenotype resulting from overexpression of the catalytic subunit of katanin. In addition, the increased stability of cortical microtubules seen upon mutation of several adjacent glutamates that serve as acceptor sites for polymodifications in Tetrahymena can be phenocopied by a knockout of the katanin gene. However, the relationship between PTMs and microtubule severing proteins could be mutual as mice with a mutation in spastin displayed axonal swellings with increased density of microtubules that were excessively detyrosinated. Interestingly, the swellings showed signs of impairment in retrograde (but not anterograde) axonal transport. It is therefore possible that lack of spastin severing activity decreases the turnover of microtubules which in turn leads to excessive modifications on microtubules. Recent studies in Drosophila support this model as a restricted knockdown of spastin in the nervous system caused excessive acetylation of microtubules at the neuromuscular junction and affected synaptic activity. Remarkably, synaptic defects caused by decreased or increased spastin function could be partially reversed by exposure to pharmacological agents that destabilize or stabilize microtubules, respectively. The simplest explanation for these data is that spastin activity promotes turnover of microtubules and indirectly decreases the levels of PTMs. Taken together, these studies indicate that a mutual interaction could exist between PTMs and microtubule severing factors. On one hand, microtubule severing factors could recognize preferentially modified microtubules. On the other hand, the severing activity could increase the turnover of microtubules that in turn negatively regulates all PTMs that accumulate on stable microtubules

Mitosis. Several PTMs are present on spindle and midbody microtubules but are absent from astral microtubules. Thus, tubulin PTMs may play a role in directing mitotic events including targeting of effector proteins to a subset of microtubules . In support of this, increased levels of detyrosinated tubulin seen in fibroblasts cultured from TTL null mice resulted in defects in spindle orientation and yeast cells expressing only detyrosinated a-tubulin displayed defects in nuclear positioning and spindle dynamics. In both animal and human cancers, TTL activity is often suppressed during tumor growth indicating that TTL suppression and resulting excessive tubulin detyrosination represent a strong selective advantage for proliferating transformed cells. In studies on polyglutamylation, Eddé and colleagues showed that polyglutamylase activity peaks in G2 whereas the levels of polyglutamylated tubulin peak during mitosis. In addition, microinjection of antibody GT335 that recognizes mono-and polyglutamylated tubulins caused a transient disappearance of centrioles and spindle defects. Palmitoylation occurs on Cys376 of vertebrate a-tubulin and mutation of the corresponding Cys377 residue to serine in yeast affected aspects of mitosis that involve interactions of astral microtubules with the cell cortex such as translocation of spindles to the bud. Thus, palmitoylation of astral microtubules could tether spindle microtubules to the plasma membrane.

1) http://mcb.asm.org/content/33/6/1114.full
2) http://www.cell.com/trends/cell-biology/fulltext/S0962-8924(13)00157-8
3) http://jcs.biologists.org/content/123/20/3447.full
4) http://www.sciencedirect.com/science/article/pii/S0960982214003248
5) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938957/
6) https://en.wikipedia.org/wiki/Kinesin
7) file:///E:/Desktop/apdf%20files/the%20tubulin%20code.pdf
8 ) http://www2.lbl.gov/tt/publications/1706pub.pdf
9 ) http://www.tandfonline.com/doi/pdf/10.4161/cc.6.17.4633

Microtubule polymerization

Microtubules are typically nucleated and organized by dedicated organelles called microtubule-organizing centres (MTOCs). Contained within the MTOC is another type of tubulin, γ-tubulin, which is distinct from the α- and β-subunits of the microtubules themselves. The γ-tubulin combines with several other associated proteins to form a lock washer-like structure known as the γ-tubulin ring complex" (γ-TuRC). This complex acts as a template for α/β-tubulin dimers to begin polymerization; it acts as a cap of the (−) end while microtubule growth continues away from the MTOC in the (+) direction

Because formation of a microtubule requires the interaction of many tubulin heterodimers, the concentration of tubulin subunits required for spontaneous nucleation of microtubules is very high. Microtubule nucleation, therefore, requires help from other factors. While α- and β-tubulins are the regular building blocks of microtubules, another type of tubulin, γ-tubulin  is present in much smaller amounts than α- and β-tubulin and is involved in the nucleation of microtubule growth in organisms ranging from yeasts to humans. Microtubules are generally nucleated from a specific intracellular location known as a microtubule-organizing center (MTOC) where γ-tubulin is most enriched. Nucleation in many cases depends on the γ-tubulin ring complex (γ-TuRC). Within this complex, two accessory proteins bind directly to the γ-tubulin, along with several other proteins that help create a spiral ring of γ-tubulin molecules, which serves as a template that creates a microtubule with 13 protofilaments





Microtubule nucleation by γ-tubulin complexes 1

Microtubule nucleation is regulated by the γ-tubulin ring complex (γTuRC) and related γ-tubulin complexes, providing spatial and temporal control over the initiation of microtubule growth. Recent structural work has shed light on the mechanism of γTuRC-based microtubule nucleation, confirming the long-standing hypothesis that the γTuRC functions as a microtubule template. The first crystallographic analysis of a non-γ-tubulin γTuRC component (γ-tubulin complex protein 4 (GCP4)) has resulted in a new appreciation of the relationships among all γTuRC proteins, leading to a refined model of their organization and function. The structures have also suggested an unexpected mechanism for regulating γTuRC activity via conformational modulation of the complex component GCP3. New experiments on γTuRC localization extend these insights, suggesting a direct link between its attachment at specific cellular sites and its activation.

The microtubule cytoskeleton is critically important for the spatial and temporal organization of eukaryotic cells, playing a central part in functions as diverse as intracellular transport, organelle positioning, motility, signalling and cell division. The ability to play this range of parts requires microtubules to be arranged in complex arrays that are capable of rapid reorganization. Microtubules themselves are highly dynamic polymers that switch between cycles of growth and depolymerization, and cells have various ways to manipulate the basic polymer dynamics to achieve precise control of the organization and reorganization of the microtubule cytoskeleton. Although many different mechanisms are used to regulate microtubule dynamics, at a fundamental level, the cell achieves control by manipulating the rates of microtubule assembly and microtubule catastrophe, as well as the timing and location of the nucleation events that give rise to new microtubules.

Microtubules are hollow tubes of about 250 Å in diameter that are assembled from α-tubulin–β-tubulin (αβ-tubulin) heterodimers in a GTP-dependent manner (Fig. below). The tubulin subunits make two types of filament contacts: longitudinal contacts run the length of the microtubule forming protofilaments, and lateral contacts between protofilaments (generally α-tubulin to α-tubulin and β-tubulin to β-tubulin) form the circumference of the microtubule (Fig. a). Microtubule geometry is not fixed, however; the more-flexible lateral contacts can accommodate between 11 and 16 protofilaments, yielding microtubules of different diameter when assembled in vitro from purified tubulin. In vivo, though, almost all microtubules have 13 protofilaments, suggesting that one level of cellular control involves defining unique microtubule geometry.


Question: How was this control achieved by natural processes, without intelligence involved ? trial and error ?

The 13-fold symmetry is probably preferred because it is the only geometry in which protofilaments run straight along the microtubule length, as opposed to twisting around the microtubule, which allows processively tracking motor proteins to always remain on the same face of the structure. An unusual feature of 13-protofilament microtubules is that, as a consequence of their helical symmetry, a 'seam' is formed from lateral α-tubulin–β-tubulin interactions, which are generally presumed to be weaker than α-tubulin–α-tubulin or β-tubulin–β-tubulin lateral contacts. The mechanism by which cells ensure 13-protofilament geometry has long been a mystery.



a | The α-tubulin–β-tubulin (αβ-tubulin) heterodimer is the fundamental repeating subunit of microtubules. When bound to GTP (shown in orange in the left panel), heterodimers come together through two types of contacts (indicated by double-headed arrows): GTP-mediated longitudinal contacts between α-tubulin and β-tubulin that form protofilaments, and lateral α-tubulin–α-tubulin and β-tubulin–β-tubulin contacts that form between protofilaments. The addition of tubulin subunits to this lattice yields the hollow microtubule. In 13-protofilament microtubules, a 'seam' is formed as a result of lateral α-tubulin–β-tubulin interactions
b | Spontaneous microtubule growth in vitro occurs in two stages: a relatively slow phase through unstable early assembly intermediates, and a rapid elongation phase. In early steps, the assembly energetics favour disassembly over assembly but, after a sufficiently large oligomer is formed by a variable number of steps (denoted here by N), assembly is energetically favoured and elongation proceeds rapidly. Whether disassembly or assembly is favoured by the assembly energetics is indicated by a bold arrow. In vivo, preformed nuclei allow microtubule growth to bypass the slow phase, providing spatial and temporal control over new microtubule growth.
c | In bulk assembly assays, the presence of a nucleator causes rapid microtubule polymerization, bypassing the lag phase observed during spontaneous growth.

Another key difference between microtubule assembly in vivo and in vitro is with regard to how new microtubules are initiated. In vitro, microtubule growth must proceed through small early assembly intermediates, for which disassembly is energetically favoured over assembly, resulting in slow initial growth. After a sufficiently large oligomer has been achieved, microtubule growth becomes energetically favourable and the addition of tubulin heterodimers proceeds rapidly (Fig. b). Significantly, rather than relying on the spontaneous initiation of new microtubules, cells have  specialized nucleation sites  that bypass the early, slower growth phase. These nucleation sites are largely found at microtubule-organizing centres (MTOCs).

Microtubules Originate from Microtubule-Organizing Centers Within the Cell

Microtubules commonly originate from a structure in the cell called a microtubule-organizing center (MTOC). An MTOC serves as a site at which MT assembly is initiated and acts as an anchor for one end of these MTs. Many cells during interphase have an MTOC called the centrosome that is positioned near the nucleus. The centrosome in an animal cell is normally associated with two centrioles surrounded by a diffuse granular material known as pericentriolar material (Figure a below).



In electron micrographs of the centrosome, MTs originate from the pericentriolar material (Figure b). The symmetrical structure of centrioles is remarkable: The walls of centrioles are formed by nine pairs of triplet microtubules (Figure a). In most cases, centrioles are oriented at right angles to one another; the significance of this arrangement is still unknown. Centrioles are known to be involved in the formation of basal bodies, which are important for the formation of cilia and flagella . The role of centrioles in non-ciliated cells is less clear. In animal cells, centrioles may serve to recruit pericentriolar material to the centrosome, which then nucleates growth of microtubules. When centrioles are missing from many animal cells, microtubule-nucleating material disperses, and the MTOC disappears. Cells lacking centrioles can still divide, probably because chromosomes can organize microtubules to some extent on their own. However, the resulting spindles are poorly organized. In contrast to animal cells, the cells of higher plants lack centrioles; their absence indicates that centrioles are not essential for the formation of MTOCs. Large, ring-shaped protein complexes in the centrosome contain another type of tubulin, -tubulin. In conjunction with a number of other proteins called GRiPs (gamma tubulin ring proteins), rings of -tubulin can be seen at the base of MTs that emerge from the centrosome. These -tubulin ring complexes ( -TuRCs) serve to nucleate the assembly of new MTs away from the centrosome. The importance of -TuRCs has been demonstrated by depleting cells of -tubulin or other components of the -TuRC; in the absence of these proteins, centrosomes can no longer nucleate MTs. In addition to the centrosome, some types of cells have other MTOCs. For example, the basal body at the base of each cilium in ciliated cells also serves as an MTOC. During cell division, centrosomes are duplicated, creating new MTOCs for each of the daughter cells.

MTOCs Organize and Polarize the Microtubules Within Cells

MTOCs play important roles in controlling the organization of microtubules in cells. The most important aspect of this role is probably the MTOC’s ability to nucleate and anchor MTs. Because of this ability, MTs extend out from an MTOC toward the periphery of the cell. Furthermore, they grow out from an MTOC with a fixed polarity— their minus ends are anchored in the MTOC, and their plus ends extend out toward the cell membrane. The relationship between the MTOC and the distribution and polarity of MTs are shown in Figure below



The nucleating ability of MTOCs such as the centrosome has an important consequence for microtubule dynamics within cells. Since the minus ends of many MTs are anchored at the centrosome, dynamic growth and shrinkage of these MTs at the plus ends tends to occur at the periphery of cells. The MTOC also influences the number of microtubules in a cell. Each MTOC has a limited number of nucleation
and anchorage sites that seem to control how many MTs can form. However, the MT-nucleating capacity of the MTOC can be modified during certain processes such as mitosis. For example, centrosomes associated with spindle poles in mitotic cells have the highest MT-nucleating activity during prophase and metaphase .





More than a century ago, the centrosome was identified as the primary MTOC in animal cells. The centrosome, organized around a pair of centrioles, serves as the central anchor point for microtubules within the cell, defining a polar microtubule array. In fungi, the functional analogue of the centrosome is the spindle pole body, which is a large multilayered structure embedded in the nuclear envelope that nucleates microtubules on both cytoplasmic and nuclear faces. Plants, on the other hand, have no centrosome equivalent, but they nevertheless have highly organized acentrosomal microtubule arrays.

Despite the variation in MTOC morphology, all MTOCs rely on γ-tubulin, a homologue of α-tubulin and β-tubulin, for nucleating microtubules. Purification of γ-tubulin from animal and yeast cells showed it to be part of larger complexes, which can directly nucleate microtubule growth. γ-tubulin is essential for normal microtubule organization in every organism in which it has been studied, and it is nearly ubiquitous throughout the eukaryotes. Moreover, it is also involved in nucleation from non-MTOC sites within cells, such as nucleation that occurs through the chromosome-mediated nucleation pathway, and in plants, which lack centrosome-like structures, suggesting that it is critical for the initiation of all new microtubules.

The γTuSC and γTuRC nucleating complexes

Early biochemical characterization of γ-tubulin showed that it was part of larger complexes that did not include α-tubulin or β-tubulin.  γ-tubulin  is part of a  complex with at least six other proteins: γ-tubulin complex protein  (GCP2), GCP3, GCP4, GCP5, GCP6 and NEDD1. The complex had a striking ring shape in electron micrographs, leading to the name γTuRC24. The γTuRC dissociates under high salt conditions to yield a stable 300 kDa subcomplex of γ-tubulin associated with GCP2 and GCP3, which is dubbed the γTuSC29


The γ-tubulin small complex (γTuSC) is the conserved, essential core of the microtubule nucleating machinery, and it is found in nearly all eukaryotes. 
The γTuSC has two copies of γ-tubulin and one each of γ-tubulin complex protein 2 (GCP2) and GCP3 (see the figure, part a).
In many eukaryotes, multiple γTuSCs assemble with GCP4, GCP5 and GCP6 into the γ-tubulin ring complex (γTuRC) (see the figure, part b). 
Previous models of γTuRC assembly suggested that GCP4, GCP5 and GCP6 together function as a cap-like scaffold for arranging multiple γTuSCs into a distinctive ring shape. This view depicts a model with six γTuSCs (12 γ-tubulins), which would leave a gap in the template, owing to the fact that microtubules are made up of 13 protofilaments. The most widely accepted model for the mechanism of γTuRC-based nucleation, the 'template model', suggests that the γTuRC acts as a template, presenting a ring of γ-tubulins that make longitudinal contacts with α-tubulin–β-tubulin (αβ-tubulin) (see the figure, part c). 
By contrast, the 'protofilament model' suggests that the γTuRC unfurls to present a γ-tubulin protofilament, which would nucleate through lateral contacts with αβ-tubulin (see the figure, part d).
A complete list of proteins that are thought to be part of the γTuSC and the γTuRC, including the more-recently identified proteins mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) and MOZART2, is given, along with alternative names for each protein (see the figure, part e). 
The five GCPs share regions of homology, although with very low levels of sequence identity (as low as 15% identity between GCP family members). Two homologous regions of GCPs, GRIP1 and GRIP2, initially defined their homology (see the figure, part f). 
Regions of more-distant homology were later shown to be more widely dispersed in the GCP sequences (green shading in part f of the figure).

The γTuSC is the core of the nucleating machinery, sufficient in itself for proper microtubule organization.

Importantly, purified γTuSC has a much lower microtubule-nucleating activity than intact γTuRC29, suggesting that the assembly state of γ-tubulin is important in determining its activity.

Early biochemical characterization of γ-tubulin showed that it was part of larger complexes that did not include α-tubulin or β-tubulin. When γ-tubulin was purified from Drosophila melanogaster embryos or Xenopus laevis eggs, it was found to be part of a ~2.2 MDa complex with at least six other proteins: γ-tubulin complex protein 2 (GCP2), GCP3, GCP4, GCP5, GCP6 and NEDD1. The complex had a striking ring shape in electron micrographs, leading to the name γTuRC24. The γTuRC dissociates under high salt conditions to yield a stable 300 kDa subcomplex of γ-tubulin associated with GCP2 and GCP3, which is dubbed the γTuSC29 (Box 1). Importantly, purified γTuSC has a much lower microtubule-nucleating activity than intact γTuRC29, suggesting that the assembly state of γ-tubulin is important in determining its activity.



a | The 8 Å cryo-electron microscopy (EM) structure of Saccharomyces cerevisiae γ-tubulin small complex (γTuSC) bound to the attachment factor spindle pole body component 110 (Spc110) is shown. This γTuSC is a single subunit of a large γTuSC oligomer (see panel b). In this view, the amino termini of γ-tubulin complex protein 2 (GCP2) and GCP3 are at the bottom, with their carboxy-terminal domains near the top interacting with γ-tubulin. In the structure, the two γ-tubulins are held apart from each other in a configuration that is incompatible with the microtubule lattice, which partially explains the relatively low nucleating capacity of free γTuSC relative to that of the γ-tubulin ring complex (γTuRC). b | Top-down and side views of the γTuSC ring are shown. The ring has six and a half γTuSCs per turn, which arise owing to a half-γTuSC overlap between the first and seventh subunits in the ring (see side view). This yields 13 γ-tubulins per turn, matching the in vivo microtubule protofilament number. The conformation of the γTuSC is unchanged in the ring structure, such that the intra-γTuSC gap between γ-tubulins remains. However, microtubule-like lateral interactions are observed between γ-tubulins at the inter-γTuSC interface.
c | The low-resolution negative-stain EM reconstruction of a single Drosophila melanogaster γTuRC (top) closely resembles the γTuSC ring shown in panel b, rendered here at lower resolution for comparison (bottom). The region of the γTuRC originally interpreted as a GCP4–GCP5–GCP6 cap is indicated with an arrow; this region appears to correspond to the N-terminal regions of GCP2 and GCP3 instead.
d | Comparison of γ-tubulin positions in γTuSC rings and α-tubulin–β-tubulin in the microtubule shows a mismatch in geometry, with alternating contacts and gaps in the γ-tubulin arrangement.

The most striking feature of the γTuSC oligomer structure is that there are six and a half γTuSCs per helical turn, owing to a half-subunit overlap between the first and seventh subunits (Fig. b). This gives 13 γ-tubulins per turn, matching the  microtubule protofilament number, with a helical pitch that is very similar to that of a microtubule. There is remarkable similarity between a single ring of the γTuSC and the low-resolution structure of the γTuRC, strongly suggesting that γTuSC assemblies like these constitute the core of the γTuRC (Fig. c). This finding also resolved the paradox of how budding yeast efficiently nucleate microtubules with only the γTuSC — they can form γTuRC-like structures from γTuSCs alone.

This is a striking example of purposeful design of the γ-tubulin ring complex which is required to nucleate the microtubules into the right shape. There would be no function for the γ-tubulin ring complex to emerge without microtubules, since  it would have no function by its own. Furthermore, it is made of several subunits which are indispensable for proper use, that is for example the attchment factors, accessory proteins, and γ-tubulins, which constitute a irreducible γ-tubulins ring complex, made of several interlocked parts, which could not emerge by natural selection. The complex has only purposeful function when microtubules have to be asssembled. So the, γ-tubulins ring complex and microtubules are interdependent. 


The increased resolution of the γTuSC subunit structure allowed the precise orientation of each γ-tubulin to be determined. The minus ends of both γ-tubulins are buried in the interaction surface with GCP2 or GCP3, and their lateral surfaces are facing adjacent γ-tubulins. Moreover, each γ-tubulin plus end is fully exposed, strongly suggesting that this surface interacts via longitudinal contacts with the minus ends of αβ-tubulin. The combination of the γ-tubulin geometry and its orientation provides the strongest evidence to date that γ-tubulin complexes function as microtubule templates. Indeed, the γTuSC rings are likely to provide the constraint that ensures the creation of 13-protofilament microtubules in vivo. It is important to note that the 13-fold architecture of the oligomer is defined almost entirely by the conformations of, and interactions between, GCP2 and GCP3, with only minor contacts between γ-tubulins within the ring. The problem of how microtubule geometry with an odd number of protofilaments can be created from a template complex with an even number of subunits is also now resolved — the half-γTuSC overlap ensures that, at most, 13 γ-tubulins are exposed for interaction with αβ-tubulin.





a | The γ-tubulin complex protein 4 (GCP4) crystal structure is shown in two orthogonal views. In the view on the left, the five α-helical bundles (i–v), small domain, amino terminus and carboxyl terminus are labelled. The C-terminal domain, consisting of bundle iv, bundle v and the small domain, was shown to directly bind γ-tubulin.
b | Two views of the pseudo-atomic model of the γ-tubulin small complex (γTuSC) are shown. The model was generated by fitting the γ-tubulin crystal structure (gold) and the GCP4 crystal structure (blue), as a stand-in for GCP2 and GCP3, into the cryo-electron microscopy (EM) reconstruction of the γTuSC (semi-transparent surface). The model reveals interaction surfaces between complex components.
c | The model also shows the positions of the conserved GRIP1 and GRIP2 domains in GCP2 and GCP3 in the context of the full γTuSC. GRIP2 is clearly involved in γ-tubulin binding. The role of GRIP1 is more ambiguous; it forms part of the lateral contact surfaces between γTuSCs, as well as part of the faces of GCP2 and GCP3 that are exposed on the outside of the γTuSC ring. 
d | When the pseudo-atomic model from panel b is fit into the cryo-EM structure of the γTuSC ring (inset), it also reveals the surfaces of GCP2 and GCP3 that are important for oligomerization. γTuSCs interact with each other primarily through the sides of bundles i and ii. 
e | The N-terminal domains of GCP2 and GCP3 are shown making intra-γTuSC and inter-γTuSC contacts, with helical bundles i–iii labelled. Equivalent surfaces of the N-terminal domains of GCP2 and GCP3 are involved in both intra-γTuSC and inter-γTuSC interactions, indicating that a single assembly rule determines the organization of the ring structure. However, the affinities have been modulated such that the stronger intra-γTuSC interactions yield a stable complex, whereas the weaker inter-γTuSC interactions allow the assembly of γTuSCs into rings to be reversible.



a | The γ-tubulins of two adjacent γ-tubulin small complexes (γTuSCs) from the γTuSC ring are shown in a top-down view. The inter-γTuSC contact is the same as a microtubule lateral contact, but the intra-γTuSC arrangement does not match the microtubule lattice. Arrows indicate the approximate motions that would align the intra-γTuSC contacts to match the microtubule lattice.
b | The negative-stain electron microscopy reconstruction of free γTuSC revealed flexibility at a hinge point in γ-tubulin complex protein 3 (GCP3), resulting in varying distances between the two γ-tubulins. 
c | Normal mode analysis of the GCP4 crystal structure predicts flexibility at the indicated position, near the equivalent hinge point in GCP3. This suggests conservation of flexibility in the GCPs. 
d | A model for the conformational activation of the γTuSC through the straightening of GCP3. In the observed conformation, the two γ-tubulins are held apart so that they cannot both be making contacts with the microtubule. However, straightening at the GCP3 hinge point by 23˚ would close the intra-γTuSC γ-tubulin gaps, bringing all of the γ-tubulins in the ring to microtubule lattice-like spacing. 
e | In this modelled state, γ-tubulin in the ring would adopt perfect 13-protofilament microtubule geometry, serving as a potent microtubule nucleator.

The flexibility of GCP4 and GCP3 and precise 23˚ angle in order to bring all of the γ-tubulins in the ring to microtubule lattice-like spacing in order to adopt to perfect 13-protofilament microtubule geometry is strong evidence of goal oriented, purposeful design. Trial and error and a non intelligent evolutionary mechanism is not feasable to bring the structure to arise in a functional way to fit precisely to the microtubules.   



a | A conserved mechanism exists for direct γ-tubulin small complex (γTuSC) attachment to the microtubule-organizing centre (MTOC). In budding yeast, the γTuSC is attached to the nuclear face of the MTOC by spindle pole body component 110 (Spc110), which serves not only to localize the γTuSC but also to promote its assembly into rings. 
b | In organisms with complete γ-tubulin ring complexes (γTuRCs), an analogous means of γTuSC-mediated attachment must exist, as γTuSC localizes at the MTOC even when all of the γTuRC-specific components (γ-tubulin complex protein 4 (GCP4), GCP5 and GCP6; shown in green) are depleted. Redundant γTuRC-specific attachment factors may also exist at the MTOC (shown in purple). 
c | Localization of nucleating complexes at non-MTOC sites within the cell is largely dependent on the presence of all three γTuRC-specific proteins.

1) http://www.nature.com/nrm/journal/v12/n11/full/nrm3209.html

Cell biology: Bacteria's new bones  1

Long dismissed as featureless, disorganized sacks, bacteria are now revealing a multitude of elegant internal structures. Ewen Callaway investigates a new field in cell biology.





Bacteria appear to have sophisticated internal structures that give them shape, and help them grow and divide.

Nearly a decade ago Jeff Errington, a microbiologist at Newcastle University in England, was toying with a strange bacterial protein known as MreB. Take it away from microbes, and they lose their characteristic cylindrical shape. The protein's obvious role in structure and even its sequence suggested a shared ancestry with actin, a protein that produces vast, fibrous networks in complex cells, forming the framework of their internal structure, or cytoskeleton. But no one had ever seen MreB in action under the microscope until Errington found just the right combination of fluorescent labels and fixatives.
In a 2001 paper, he presented MreB (orange in the illustration) fluorescing brilliantly and painting barbershop-pole stripes around the rod-shaped bacterium Bacillus subtilis1. “We got these amazing pictures. It was one of those few times in a scientific career when you do an experiment that completely changes your way of thinking,” says Errington.
For more than a century, cell biology had been practised on 'proper' cells — those of the eukaryotes (a category that includes animals, plants, protists and fungi). The defining characteristic of eukaryotic cells is their galaxy of internal structures: from the pore-studded nucleus that contains the genome, to the fatty sacs of the Golgi, to the myriad mitochondria, and of course the networks of protein highways that ferry things around the cell and give it shape and the capacity for movement. These elements form a catalogue of cell biology's greatest discoveries, and all of them are absent in bacteria. Hundreds to thousands of times smaller than their eukaryotic cousins, and seemingly featureless, bacteria were rarely invited to the cell biology party. But Errington's discovery has been part of a movement that is changing that.
Dyche Mullins, a cell biologist at the University of California, San Francisco, had spent most of his career untangling the network of molecular cables and scaffolding that enforces order in the eukaryotic cell. With Errington's paper, Mullins saw the lowly bacterium anew. “There was a lot of organization in bacterial cells we were just missing,” he says. He has since devoted much of his time to studying them. Last month, Mullins chaired the annual meeting of the American Society for Cell Biology in Washington DC. That he was chosen for the job is a clear indication that bacteria have made it on to the guest list.
Lucy Shapiro, a microbiologist at Stanford University in California gave bacteria an hour-long tribute at the meeting. “People more or less thought the bacterial cell was a swimming pool and the chromosome was this ball of spaghetti,” says Shapiro, whom many credit for launching the field of bacterial cell biology.

External contractors

Busied by growth, propagation and little else, a bacterium's life can seem an endless cycle of fecundity. Well-fed bacteria in rich, sterile culture can divide every half hour or so. The cytoskeleton is the linchpin of efficient cell growth and division, but researchers are only starting to explain how.
Take FtsZ (illustrated in yellow), a protein that ties a belt around the belly of nearly every species of bacterium. Without FtsZ — a barely recognizable cousin of the eukaryotic protein tubulin — rod-shaped bacteria called bacilli grow longer and longer without splitting in two. Somehow FtsZ cinches the dividing cell closed, says Harold Erickson, a cell biologist at Duke University Medical Center in Durham, North Carolina. Tubulin is involved in eukaryote cell division, but its role is completely different. Microtubules, formed from tubulin, pull chromosomes apart during cell division through a process that has been studied extensively.
Erickson started out studying tubulin. But intrigued by the pictures of internal FtsZ structures coming out of other labs in the 1990s, he began reading up on FtsZ. When the time came to reapply for a grant, he devoted half of his proposal to the bacterial protein. “I decided, 'I don't have any great ideas about what to do with tubulin',” he recalls. It took a couple of applications to get funding, but Erickson hasn't looked back.




Electron microscopy suggests that Escherichia coli and other bacteria have no organized internal structure.

Squeezing two cells out of one is just one of the cytoskeleton's duties. When bacteria divide they need to resculpt a rigid cell wall built out of peptidoglycan, a polymer consisting of sugar and amino-acid bricks. Without the MreB protein wound around the shell of a bacillus, it grows spherical (see 'How bacteria get in shape'). The protein directs the construction and destruction of the cell wall, says Zemer Gitai, a microbiologist at Princeton University in New Jersey. One theory is that MreB and its relatives build a protein scaffold inside the cytoplasm that tells the cell wall's enzyme contractors outside the cytoplasm where to lay new bricks. Because two layers of membrane separate the MreB helix from the cell wall, other proteins must forge the connection, says Gitai.
Also, when a bacterium divides, each new cell must have its own DNA. Most of a bacterium's thousand or so genes sit on a long chromosome, but smaller rings of DNA called plasmids also help a cell by supplying antibiotic resistance and other perks.
Mullins's lab studies a bacterial version of actin, called ParM, which ensures that as a cell splits in two, each receives a copy of a specific plasmid. Without the protein, many cells will invariably lose the plasmid and the drug resistance it provides.



Cryo-electron tomog raphy of a mutantCaulobacter shows gobs of FtsZ filaments (red) lining the constriction site as the cell tries to divide.

To avoid this fate, a strand of ParM molecules (shown in green) latches onto two freshly replicated plasmids (purple), like the chain to a pair of handcuffs. The two circles start close to one another, but as more ParM molecules leap onto the chain, the plasmids spread to opposite ends of the cell. Mullins's group found that the ParM chain grows pretty much on its own — a startling contrast to our own actin, which requires other players to speed extension. Although related to actin, ParM works more like tubulin, constantly reinventing itself by adding and shedding units. “That blows my mind,” Mullins says.
His team is now looking at how other plasmids ensure their legacy, to say nothing of the bacterial chromosome, a DNA loop thousands of times longer than any individual plasmid. “We know very little. For me, the most important unanswered question in cell biology is how bacteria segregate their chromosomes,” says Mullins.
The wealth of questions and dearth of answers makes the field very attractive. Every time a new bacterium is sequenced, researchers have the opportunity to find new structural elements, often with surprising roles. One of the latest additions is an actin protein, MamK, found in bacteria endowed with iron-containing structures called magnetosomes. By sensing Earth's magnetic tug, the bacteria can position themselves in the environment best suited to their needs. For the compass to work, a cell's dozen or so magnetosomes need to line up in a row, and MamK forms their track². Arash Komeili, a microbiologist at the University of California, Berkeley who first identified the protein's role says that by scouring genome databases he has found genes similar to MamK in bacteria with no magnetosomes.

Seeing is believing

Although bacterial cell biologists such as Komeili can use genomics to hunt for new features of the cytoskeleton, pictures make a stronger case, he says. Advances in optics and microscopy are one reason the bacterial cell is only now getting its dues. At a few micrometres, bacteria are often not much longer than the limits of a light microscope, so even the best lens in the world won't bring any detail to a molecular cable a few nanometres thick.


An actin-like filament called MamK (yellow) organizes a chain of magnetosomes (iron-containing structures) in the magnetic bacteriumMagnetospirillum magnetotacticum.

Peering deeper into a bacterial cell requires abandoning the light waves that obscure detail. Electrons, which have a far shorter wavelength than visible light, provide staggering insights into eukaryotic cell structure, such as the ribosome-studded endoplasmic reticulum or the perfectly arranged bundle of microtubules that build a cilium tail. In bacteria, the same electrons paint a blurry mush. Even the most recent edition of the hallowed text Molecular Biology of the Cell sees bacteria under the magnification of an electron microscope as chaotic vessels: “This cell interior appears as a matrix of varying texture without any obvious organized internal structure,” the authors write.
A more promising technology — cryo-electron tomography — might be the answer. Instead of coating cells with gold or dousing them in harsh fixatives, cryo-EM, as it is often called, takes pictures of flash-frozen samples. “We're looking at cells in a nearly native state,” says Grant Jensen, a biologist at California Institute of Technology in Pasadena. The gentle treatment keeps the bacterial cytoskeleton intact. “If you thawed them out, most of them would probably swim away.”
Cryo-EM has the added benefit of allowing researchers to combine numerous angles of a cell into a three-dimensional picture, just like a computed tomography scan does. Recently, Jensen's lab collected images of rings of FtsZ lining the insides of a bacterium calledCaulobacter and pinching its membrane — a model predicted by others but never seen before.
When early searches for bacterial genes resembling eukaryote scaffold-protein genes found nothing, scientists assumed that these proteins evolved after bacteria split from eukaryotes, some 1.5 billion to 2 billion years ago. The discovery of the bacterial cytoskeleton has turned that conclusion on its head.
FtsZ may be the great-grandfather of cell division, says Erickson, whose lab recently showed that the protein makes rings inside microscopic droplets of oil, a stand-in for early life. Although cell division now is an elaborate choreography between dozens of players, the earliest cells may have needed just FtsZ to split in two. Erickson points out that the protein contains none of the amino acids, such as tryptophan and arginine, that some believe only to have shown up later in evolution.
As cytoskeletons evolved, they took on new chores and snowballed in complexity. At some stage after eukaryotes branched off from bacteria, the eukaryote cytoskeleton seems to have frozen in time. From yeast through to people, its proteins do many of the same jobs, such as towing sister chromosomes to opposite ends of a dividing cell or making sure the endoplasmic reticulum nestles up against the nucleus. More complex eukaryotes might use actin to flex muscles and keratin to make hair, but those tasks are variations on a theme.
Not so with bacteria, says Mullins. Actins that determine cell shape work differently across the bacterial world, and some rod-shaped bacteria, such as tuberculosis, don't even have them. Due to their vast numbers and unicellular lifestyle, “bacteria can play around with fundamental mechanisms for doing things in a way that eukaryotes can't”, he says.
But the shared trait of bacterial and eukaryotic cytoskeleton proteins — self assembly — means that bacteria can shed light on the workings of more complex species. For example, the molecular structure of MreB explained how actin molecules stick together. And in most cases, bacterial proteins yield to laboratory tinkering with less resistance than the eukaryotic kind. Turning up the expression of actin, for instance, kills many eukaryotic cells, but bacteria don't seem to mind.
And bacteria, because they have few genes, are ideal for addressing fundamental questions about all cellular life. Although cytoskeletons seem to act as organizing centres in bacteria and eukaryotes, no one yet understands how these proteins travel to precise spots in a cell, to one end or the other or to the site where one cell splits in two.

As well as being intellectually stimulating, probing the insides of bacteria has practical applications, and bacterial cell biologists recognize the need to remind funding agencies such as the National Institutes of Health of that. For example, a chemical named A22 slows bacterial growth by stopping MreB from forming into long cables, and without FtsZ many bacteria will die. No antibiotics yet target the bacterial cytoskeleton, but with drug resistance on the rise, structures such as the MreB helix and the FtsZ ring could prove to be chinks in the bacterial armour.
But as researchers struggle to piece together the bacterial cell, cures for disease are far from the minds of most. For Mullins, the field's progress has vindicated his dive into the bacterial swimming pool, although he and others still haven't come close to its deep end. “There's a lot of unexplored biology,” he says.

further readings :

1) http://www.nature.com/news/2008/080109/full/451124a.html

http://www.hhmi.org/research/structural-studies-macromolecular-assemblies

Scientists unravel the mystery of the tubulin code 1





 TTLL7 (GOLD STRUCTURES ON THE LEFT) IMPACTS CELL FUNCTION BY BINDING TO MICROTUBULES (SILVER STRUCTURE MADE UP OF PURPLE AND YELLOW SUBUNITS) AND ADDING CHEMICAL MARKERS TO THE SURFACE. view more 

Driving down the highway, you encounter ever-changing signs -- speed limits, exits, food and gas options. Seeing these roadside markers may cause you to slow down, change lanes or start thinking about lunch. In a similar way, cellular structures called microtubules are tagged with a variety of chemical markers that can influence cell functions. 
The pattern of these markers makes up the "tubulin code" and according to a paper published in Cell, scientists at NIH's National Institute of Neurological Disorders and Stroke (NINDS) have uncovered the mechanism behind one of the main writers of this code, tubulin tyrosine ligase-7 (TTLL7).

"Understanding the structural characteristics of this specific molecule opens the door to learning how elaborate patterns of chemical markers are laid down on microtubules. Deciphering the tubulin code could tell us how the markers affect normal cellular function as well as what happens when they are damaged, which can lead to neurodegenerative disorders," said Antonina Roll-Mecak, Ph.D., NINDS scientist and senior author of the study.



TTLL7 is a protein that adds glutamate tags onto microtubules. Using a number of advanced imaging and biochemical techniques, Dr. Roll-Mecak and her colleagues at the Scripps Research Institute in La Jolla, California, have revealed the 3-D structure of TTLL7 bound to the microtubule. There are nine proteins that make up the TTLL family, but TTLL7 is the most abundant in the brain and one of the main tubulin code writers. These results represent the first atomic structure of any member of the TTLL family.
The findings define how TTLL7 interacts with microtubules and how members of the TTLL family use common strategies to mark microtubules with glutamate tags. Dr. Roll-Mecak and her team were able to see how TTLL7 positions itself on the microtubule by grabbing onto the microtubule tails.

"This was a very surprising result, as no one had been able to visualize these tails on the microtubule before," said Dr. Roll-Mecak.
Microtubules are cylindrical structures that provide shape to cells and act as conveyor belts, ferrying molecular cargo throughout cells. Although all microtubules have the same basic appearance, they are marked on their outside surface with a variety of chemical groups. These markers impact a cell's activity by changing the stability of microtubules, thus affecting cell shape, or by repositioning molecular cargo traveling on the microtubules.

"The microtubule markers are constantly being added and removed, depending on the local needs of the cell. Think about a highway system where street signs are constantly changing and roads are quickly built or torn apart," said Dr. Roll-Mecak.

The most common microtubule marker in the brain is glutamate. The addition of glutamate markers to microtubules plays important roles in brain development and brain cell repair following injury. For example, one of the signatures of damaged cells in cancer or blunt trauma is a change in the pattern of these microtubule markers. In addition, mutations in TTLL genes have been linked with several neurodegenerative disorders.

"Our detailed analysis of TTLL7 also may provide important insights into ways that the other members of the TTLL family function. This study is the first step in gaining a more complete picture of how the tubulin code is established," said Dr. Roll-Mecak.
Her lab plans to extend their research by investigating interactions between members of this family of proteins. "We want to mix and match the TTLL proteins to see how we can control patterns of microtubule tagging. From that, we can learn how the cell is making those patterns and what happens during cellular damage, as in cancers or neurodegeneration, when these patterns are disrupted," said Dr. Roll-Mecak.
She added that this research may lead to the development of small molecules that can regulate activity of TTLL proteins, which may have implications for disorders linked to mutations in TTLL genes.

1) http://ekaweb01.eurekalert.org/pub_releases/2015-05/nion-sut051215.php

High-Resolution Microtubule Structures Reveal the Structural Transitions in αβ-Tubulin upon GTP Hydrolysis 1

Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice. . We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region.

Microtubules are ubiquitous cytoskeletal filaments critical for multiple cellular processes, including intracellular trafficking, establishment and maintenance of cell morphology, and cell division (Hyams and Lloyd, 1993). For many microtubule-dependent processes, the underlying dynamics of the polymer play a pivotal role. Perhaps the most striking example is mitosis, when chromosome motions are driven by microtubule dynamics and segregation is primarily powered by microtubule depolymerization (Desai and Mitchison, 1997, McIntosh et al., 2010 and Rieder and Salmon, 1994). Highlighting this fact, many successful antiproliferative drugs bind to tubulin and interfere with microtubule dynamics (Dumontet and Jordan, 2010). Describing the conformational cycle accompanying tubulin polymerization, nucleotide hydrolysis, and microtubule depolymerization is essential for our understanding of microtubule dynamics


Dynamic instability, the stochastic switching between phases of microtubule growth and shrinkage, is driven by the binding and hydrolysis of GTP by the αβ-tubulin dimer (Mitchison and Kirschner, 1984). Tubulin dimers associate longitudinally to form polar protofilaments, which associate laterally to form a tube. Subunit addition occurs preferentially at the end of the microtubule capped by β-tubulin subunits, termed the “plus end.” αβ-tubulin contains two GTP-binding sites (Figure 1A). The N-site (nonexchangeable) in α-tubulin is buried within the tubulin dimer at a longitudinal monomer-monomer (or intradimer) interface (Nogales et al., 1998). This site is constitutively occupied by GTP and has been ascribed a structural role (Menéndez et al., 1998). The nucleotide at the E-site (exchangeable) in β-tubulin is exposed on the surface of an unpolymerized dimer and the terminal subunits of a microtubule plus end (Mitchison, 1993 and Nogales, 2000). Free αβ-tubulin dimers exchange bound guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the E-site, rendering them competent for polymerization (Figure 1A). Upon addition of a tubulin dimer to a growing microtubule plus end, the α-tubulin subunit in the incoming dimer contacts the E-site GTP of the terminal β-tubulin subunit, completing the binding pocket that enables hydrolysis (Nogales et al., 1999). Thus, microtubule growth and GTP hydrolysis are coupled, giving rise to the metastable character of this polymer. Whereas a lattice of GTP-tubulin is stable and promotes polymerization, the GDP-tubulin lattice is unstable and prone to depolymerization, or “catastrophe” (Desai and Mitchison, 1997). In the long-standing “GTP-cap” model (Mitchison and Kirschner, 1984), a microtubule will continue to grow as long as it contains GTP-tubulin subunits at its plus end (i.e., subunit addition outpaces hydrolysis). When this GTP cap is lost, rapid depolymerization ensues (Figure 1B).






Figure 1.  High-Resolution Cryo-EM Structures of Dynamic and Stabilized Microtubules
(A) Cartoon of the αβ-tubulin dimer, which spontaneously exchanges bound GDP for GTP in solution.
(B) Cartoon illustrating structural intermediates of microtubule polymerization and depolymerization.
(C) Cryo-EM maps of GMPCPP (left panel, 4.7 Å resolution), GDP (middle panel, 4.9 Å resolution), and Taxol-stabilized (right panel, 5.6 Å resolution) microtubules, viewed from inside the microtubule lumen. α-tubulin, green; β-tubulin, blue; GMPCPP/GTP; orange; GDP, pink; Taxol, yellow. 

The detailed molecular mechanism by which tubulin GTP binding and hydrolysis controls microtubule dynamics remains elusive despite decades of intensive study. Structural studies have led to the consensus view that conformational changes in tubulin must be correlated with the transition from polymerization to depolymerization. A straight tubulin conformation is found within the body of the microtubule (Li et al., 2002 and Nogales et al., 1999), and all high-resolution structural analyses of this state to date have been limited to electron crystallography of zinc-induced two-dimensional (2D) sheets, which contain protofilament-like head-to-tail assemblies of straight αβ-tubulin (Nettles et al., 2004 and Nogales et al., 1998). A curved conformation is found in microtubule depolymerization peels (Mandelkow et al., 1991), head-to-tail arrays of bent tubulin heterodimers wherein longitudinal contacts are maintained after lateral contacts are broken (Figure 1B). The existence of peels as a depolymerization intermediate, together with the fact that contacts between protofilaments are mediated by a series of loop-loop interactions (Li et al., 2002 and Sui and Downing, 2010), has led to the assumption that lateral contacts are the most labile of the microtubule lattice interactions. In this model, hydrolysis-induced destabilization of the distant lateral contacts would occur through an unknown allosteric mechanism.

1) http://www.sciencedirect.com/science/article/pii/S0092867414004838

Intracellular Spatial Localization Regulated by the Microtubule Network 1

The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a “structured cytoplasm”, in contrast to a free and fluid environment.

The microtubule network is commonly recognized as the major mechanical skeleton that drives cell division. Numerous seminal experimental observations led us to speculate that the microtubule network could also serve as a spatial regulator for cellular components. Many molecules can bind with microtubules either directly, or indirectly through other microtubule-binding molecules. Binding to microtubules causes the partial sequestration of the molecules by the microtubule network, the degree of which depends on the binding affinities, as well as the microtubule density. In addition, motor protein-mediated binding leads to convective fluxes that help rearrange the spatial localization of the cargo molecules. For example, in pace with the progression of mitosis, a number of mitotic spindle checkpoint proteins accumulate at the poles of the mitotic spindle via the active transport of dyneins along the microtubules . Microtubule-mediated spatial regulation also plays critical functional roles in various biological processes, e.g. the determination of embryo polarity and cell fates in the syncytial Drosophila embryo, the establishment of dorsal-ventral axis in the Xenopus embryo , the asymmetric cell division in the Drosophila central brain neuroblast.




1) http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034919

The chemical complexity of cellular microtubules: tubulin post-translational modification enzymes and their roles in tuning microtubule functions 1

Poly-glutamylation and poly-glycylation are not simple ON/OFF signals like tyrosination/detyrosination, phosphorylation or acetylation. The specific length of the added chain shows large variation: glutamic acid chains can be as long as 20 residues, although most are between one and six

Unless you get a specific amino acid combination and size, as for example 20 residues mentioned above, there is no function. To get these 20 residues, a specific genetic instruction is required. Any mutation that does not provide the right combination and size will not provide any function. If a gene does not contain " molecular knowledge ", then it has no function, it confers no selective advantage. Thus, before a region of DNA contains the requisite molecular knowledge, natural selection plays no role in guiding its evolution. Chance controls which mutations survive. Thus, molecular knowledge can be related to a probability of evolution. 2


1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459347/
2) http://reasonandscience.heavenforum.org/t2062-proteins-how-they-provide-striking-evidence-of-design#4055

CYTOSKELETAL ARRAYS

Darwins doubt, page 209

Eukaryotic cells have internal skeletons to give them shape and stability. These "cytoskeletons" are  made of several different kinds of filaments including those called the "microtubules." The structure and location of the microtubules in the cytoskeleton influence the patterning and development of embryos. Microtubule "arrays" within embryonic cells help to distribute essential proteins used during development to specific locations in these cells. Once delivered, these proteins perform functions critical to development, but they can only do so if they are delivered to their correct locations with the help of preexisting, precisely structured microtubule or cytoskeletal arrays . Thus, the precise arrangement of microtubules in the cytoskeleton constitutes a form of critical structural information. These microtubule arrays are made of proteins called tubulin, which are gene products. Nevertheless, like bricks that can be used to assemble many different structures, the tubulin proteins in the cell's microtubules are identical to one another. Thus, neither the tubulin subunits, nor the genes that produce them, account for the differences in the shape of the microtubule arrays that distinguish different kinds of embryos and developmental pathways. Instead, the structure of the microtubule array itself is, once again, determined by the location and arrangement of its subunits, not the properties of the subunits themselves. Jonathan Wells explains it this way: "What matters in [embryological] development is the shape and location of microtubule arrays, and the shape and
location of a microtubule array is not determined by its units." For this reason, as University of Colorado cell biologist Franklin Harold notes, it is impossible to predict the structure of the  cytoskeleton of the cell from the characteristics of the protein constituents that form that structure.Another cell structure influences the arrangement of the microtubule arrays and thus the precise structures they form and the functions they perform. In an animal cell, that structure is called the centrosome (literally, "central body"), a microscopic organelle that sits next to the nucleus between cell divisions in an undividing cell. Emanating from the centrosome is the microtubule array that gives a cell its three-dimensional shape and provides internal tracks for the directed transport of organelles and essential molecules to and from the nucleus. During cell division the centrosome  duplicates itself. The two centrosomes form the poles of the cell-division apparatus, and each daughter cell inherits one of the centrosomes; yet the centrosome contains no DNA. Though
centrosomes are made of proteins—gene products—the centrosome structure is not determined by genes alone.

The Enormous Complexity of Transport Along the Axon

Some scientists consider scaffolding fibers and tubules in the neuron to be the seat of consciousness. They respond instantly to any mental event with massive movement and construction—building and rebuilding the structures for dendrite spines and axon boutons at synapses in the ever-changing neuron.

Microtubules are the critical highways for materials, mitochondria and vesicles along the vast length of the axon. Many neuro-degenerative diseases can be traced to dysfunction of microtubules. In fact, the cause of Alzheimer’s might be the disintegration of tau molecules that provide strength and stability to the microtubule structures.

Scientists are amazed that microscopic materials can be transported more than several feet along one neuron that goes from the spinal cord to the foot. This is equivalent scale to a person carrying a package walking along the wall of China.


Now, research is showing many different elaborate motors and hundreds of adaptors and factors utilized in this transport. In fact, the extremely complex mechanisms are different for each type of cargo—messenger RNA, small molecules, vesicles filled with neurotrophins, mitochondria, ribosomes, and huge organelles like lysosomes and phagosomes. Each cargo is tagged for its destination. Also, each section of the axon—the initial segment and regions far from the cell body—have different types of transport regulation. Research is showing the enormous complexity of transport along the axon.

Three Types of Tubules and Many Motors for Each

While there are three basic types of scaffolding tubules—actin, intermediate filaments, and the larger microtubules—it is the microtubules that provide transport along the axon. A previous post discussed these three tubules and another discussed the important myosin motors that work with actin and are critical for neuroplasticity.


There are thousands of different types of lattice scaffolding structures in human cells—each built with these three molecules. Actin builds a membrane’s moving edge for a growing axon or dendrite. Microfilaments are the most flexible that make strong connections by elaborate branching. Microtubules provide stable structure for transportation.

Microtubules Build Structures Using Many Other Molecules

Microtubules are the strongest and most elaborate tubules. They are used for major transportation traffic as well as complicated scaffolding at all levels of neuronal function. They, like the other tubules, are critical for neuroplasticity. Microtubules have hundreds of helper molecules that are needed in different situations.


The microtubule is built as a spiral cylinder with a positive charge on the growing leading edge and a minus charge on the other. Transport away from the cell body carries lipids, proteins, energy producing mitochondria, vesicles of all types and other materials for the synapse. Transport back to the cell body is critical for mitochondria going back and forth, removal of debris in vesicles and signals related to damage of the distant axon regions. In fact, defects in the ability to transport debris might be the primary cause of Alzheimer’s disorder.

Microtubules make a reliable shape for a track where motors shuffle or step along carrying many kinds of loads. There are many motors that use these tracks. But, the two major ones in the neuron are the protein motor kinesin, which moves away from the cell center toward the synapse and the protein motor dynein, which moves material toward the cell center.


Rapid growth of axons occurs with many parallel microtubules enlarging at the plus end. The organization of microtubules is much more variable and complex in the smaller, but equally rapidly growing dendrites. For the dendrite, the minus end near the cell body is either connected to the centrosome (an organelle that serves as the main microtubule organizing center) or they are capped for stability.

There are a host of associated proteins that maintain stability. A famous one is tau that holds these arrays of microtubules together until they collapse in Alzheimer’s and form neurofibrillary tangles (see post). There are, however, many other proteins associated with microtubules that, when defective, produce disease. These include many different scaffolding proteins, motors and adaptors used to transport different material. These associated proteins can, even, regulate the specific motors on the microtubule.


There is one group of proteins that interact with the growing positive tip of the microtubule, called +TIPs (plus end interacting proteins). Taking care of the growing tips is a particularly difficult job because tips are unstable and always changing in different ways—growing, rapidly shortening, dramatically breaking and being rescued.

Kinesin and Dynein Motors

Kinesins are the motors that travel from the cell body to the synapse.Dyneins go back to the cell body. They were both discovered thirty years ago and operate with a stepping motion as if they are walking carrying a bundle.

Kinesin Motors

From Moez wik
There are 45 genes that regulate and manufacture Kinesin—brain cells utilize 38 of these. The neuron is unique in its complex usage of these motors and their microtubules, so there is a much greater regulation than in other more stable cells. There are many versions of the three basic kinesins—kinesin1, 2 and 3.
Kinesin-1 takes proteins, organelles, RNA and vesicles at a rate of around 1um/second (a micrometer is 0.000001 of a meter). The structure includes two heavy subunits and two light subunits. The light chains are involved in mechanisms for stopping the transport. They move toward the plus growing end of the microtubule. Each step is 8 nm long (a nanometer is 0.000000001 of a meter). This step has a strong attachment and force and can win a match with an opposing motor. But, they still can detach when it is too difficult. New proteins are transported very rapidly. Organelles have been clocked with movement of 400 mm/day (which is 1um/second).

The moving part of Kinesin-2 is either a single molecule or made of multiple subunits. It specializes in carrying material for membranes. The attachment is not as strong as K1 and can detach if presented with a tug or war from an opposing motor.


Kinesin-3 is altered based on what it is carrying and can be made to travel much faster when carrying critical organelles. K3 carry sacs with neurotransmitters and materials for the synapse. These can operate with increased force because of the importance of their cargo that can overcome opposing transport and obstacles.

Dynein Motors

Unlike kinesin, the active dynein motor subunit is made from one gene. The intermediate and light chains have two genes each. It, also, has heavy and light chains for different structures used in carrying different loads. These subunits can self assemble into different arrangements for different situations.

Dynein travels very fast, but can, also, take back and side steps, unlike kinesins. It is not as strong as kinesin and loses direct battles with it when traveling in opposite directions along the same microtubule. But, its sideways movement makes it very effective in overcoming obstacles. Dynein can work in teams of motors for larger more complex cargo.


Dynein, most of the time, needs a factor called dynactin, a very complex molecule made of several proteins that activates dynein for use. It is critical for the function of neurons by binding to the microtubule and the dynein motor. Dynactin has many different binding sites and has a wide range of activity including special functions in the initial axon region and other distant regions.


Motors In Competition – How Are They Regulated
Many cargoes use multiple motors at once, sometimes as many as a dozen. Heavy or large cargoes, such as lysosomes, have many attached motors for transport that work together. These teams of motors can consist of kinesin-1, kinesin-2, and dynein together. Kinesin 1 and 2 work together to transport vesicles with prions inside. Autophagosomes (for cleaning debris) travel a long way on the axon with dynein and kinesin working together.



How are all these different motors regulated? Is each regulated or is there overall direction; or are the motors competing? One model says kinesin is strongly regulated and less for dynein. However, very new research finds that in many cases, the scaffolding molecules regulate this tug of war. (Another finding that supports consciousness in these molecules).
Each type of cargo, motor and adaptor seems to be regulated differently.There is reason to think that for lysosomes there might be a tug of war between kinesin and dynein. For autophagosomes the dynein is dynamically lowered by the microtubule so that kinesin can push it forward.

Kinesein-1 has been shown, at times, to regulate itself and change its speed and force. The inhibition occurs with factors that bind to kinesin’s tail where it connects with the motor. Scaffolding proteins alter this inhibition, which can cause movement in either direction.


There are, in fact, many factors that alter movement in different ways with different cargoes. Dynein regulation can be caused by attachment and detachment of ATP energy molecules, which alters its force. This causes the motor to attach more strongly to the microtubule with less movement. Another factor, the protein huntingtin, regulates vesicles with BDNF and autophagosomes, and is relevant to Huntington’s disease. There are many enzymes involved in this process.

Transport Regulation Mechanisms Can Be General or Specific

Each large organelle takes part in the regulation of its own movement and they use quite different mechanisms. For some organelles, specific motors stay with an organelle even if the motor is not being used at that time. Specific groups of opposing motors move very large organelles. These mechanisms are very complex and include kinase enzymes, multiple molecular cascades, attaching phosphorus energy particles, and scaffolding protein activity.

Fast Forward Transport for Neurotrophins

Neuropeptides, neurotransmitters, and neurotrophins are critical to neuron function and are carried in special vesicles called dense core or granular vesicles by kinesin-3. The mechanism for granular vesicles changes the structure of the kinesin-3 subunits as well as the specific adaptors. These particular vesicles cannot be returned and new ones must be sent all the way from the cell body to the synapses. Each is tagged for delivery to specific dendrites and axon tips with different material. There are specific regulators of the movement as it approaches the target. The dynein is disabled at the site to avoid return of useless sacs. The sacs are recycled locally.

Protein that is the precursor of amyloid, APP (amyloid precursor protein), is carried in a vesicle at very rapid rates mostly away from the cell body. The mechanism includes a particular molecule that is phosphorylated to provide the switch determining direction.


BDNF (brain derived neurotropic factor) is a critical molecule for neuronal function and is transported from the cell body to the synapse. The vesicle transport, uniquely, uses huntingtin and makes a scaffolding structure with kinesin-1 and dynein motors. Huntingtin phosphorylation is the switch to go forward and the opposite dephorphorylation goes backward. (A defective huntingtin causes Hungtington’s disease)
Neurotrophins are factors that are secreted locally near the synapse and maintain the health and very life of the neuron. Without them the neuron will die. These critical molecules bind at the presynaptic terminal–the end of the axon. From there, they are transported backwards to the cell body where they stimulate networks of genes to maintain the health of the neuron. It is a signal from the tissue where the axon has landed to help keep the neuron alive.

Neurotrophins are taken into the cell in vesicles (signaling endosomes) and taken along the microtubules to the cell body. This transport is very active, fueled by ATP production along the axon from mitochondria. Mitochondria are moved (see post on microtubules alive in the cell) along the microtubules to provide this needed energy (30% of mitochondria are moving at any one time).

Transporting Mitochondria for Energy

The movement of mitochondria has unique regulation. Mitochondria are slowed near the synapse so they will stay there and provide energy. The signal to slow is the increased levels of calcium from the neuron’s action potential at very particular places near the synapse.


Those mitochondria that are nearby–15nm away–are not affected. But, if they travel through the area of increased calcium they stop. When specific dendrites are very active, the mitochondria maintain a higher level of energy production. There are many complex mechanisms of the transport motors and adaptors to provide this calcium effect.

In previous posts, microbes’ decision-making ability, communication, and group behavior were discussed. It described the process of mitochondrial fission and fusion that is highly regulated through the endoplasmic reticulum of the neuron. Mitochondria within the neuron function as a microbe community working together for the goal of providing energy throughout the very complex neuron.They communicate with each with various signals and to the cell through intermittent contact with the endoplasmic reticulum.

Transporting Endosomes and Lysosomes

Critical vesicles, such as endosomes, phagosomes and lysosomes, are carried on the transport systems, many going back and forth rapidly. Like mitochondria these are essential organelles that are needed to clean up debris or bring information to different regions. There are unique mechanisms for the transport away from the cell body using adaptors that link to the subunits of kinesin-1 and 2, and others to dynein.

Transport of Structural Proteins for Scaffolding

The transport of hundreds of different structural materials to constantly build and rebuild the structure of the axon is slower than that of the organelles. There are two different slow speeds for these materials—1 mm per day and 10 mm per day. The slower speed is for intermediate filaments and tubulin (to make microtubules). The faster rate is for a large number of smaller molecules. It is much harder for scientists to study the slow transport because of the need for very long observation of living microscopic material.


Different intermediate filament assembled units are transported by kineisin-1 and dynein. One of the subunits binds directly to the motors. The transport seems to be in small movements with rests in between. The transport of actin and microtubule structures is not as well known. It is especially difficult to study microtubules, because they are built up and broken down so rapidly. Tubulin for microtubules is transported either as two molecules or as small microtubule assemblies.
More than two hundred other molecules are transported to be used in construction. Synapsin is one critical protein that combines into complexes, which latch onto passing motors. The slow rate of transport is related to the complex attaching and reattaching with periods of travel and waiting. Despite this type of stop and go system, the slow transport provides three times the material as the fast transit. The proteins that travel from the spinal cord to the foot can take from four months to a year for transport.

Transport Control in Different Axon Regions

Each region of the axon has very different scaffolding structures and different mechanisms. 


In the initial segment there is a very specific structure with unique stabilizing molecules. This region makes sure that cargoes headed for the dendrites and the cell body don’t start travelling down the long axon. Some cargoes are targeted for the dendrite, but start down the axon and are stopped and re routed. Unique adaptors and modifiers for microtubules in this region help this process.
The distant regions of the axons direct the return of cargoes all along the axon to the cell body by unique mechanisms. The growth of microtubules in these distant regions is different. It is much more active and variable with many different plus ends appearing. There are special proteins in this region that interact with the active plus ends. Dynactin, also, interacts with the special proteins at the plus ends. These complex very active mechanisms appear to give direction to transport heading back to the cell body
Since the electrical charge seems to be related to this increased intelligent activity of the microtubules, it is has be questioned whether electrical fields are involved. Please see the post on electrical fields and gradients determining cellular behavior.

Making Proteins All Along the Axon

While many of the proteins are manufactured in the cell body and transported the long way to the synapse, there is increasing evidence that there is, also, local ribosome activity all along the axon and in the dendrites. Large ribosomes are, first, transported to distant sites. To use the local ribosomes, messenger RNA is transported to these regions.


Research has uncovered more about the production of local proteins in the dendrite. In order to transport messenger RNA they need to be altered. The usual mechanisms to find a ribosome are held in check until they travel to the distant ribosome. Specially altered messenger RNAs are put in a granule vesicle with the necessary proteins and even some large ribosomes. These specially packed vesicles go back and forth in both directions—wherever needed—and seem at times to oscillate.
The movement of messenger RNA vesicles is greatly increased when there is nerve injury, as well as by some chemical signals. When there is injury, special proteins are manufactured that bind to the motors stimulating more rapid transport of supplies. There are, also, genetic activating factors that stimulate more protein manufacturing. With nerve injury, complex multi protein structures produce transport back to the cell. They signal the activation of genetic machinery to rev up manufacturing for repair. This process involves many steps from a variety of enzymes triggered by energy rich phosphorus.

A faster signal of the injury is increased calcium at the injury site that radiates back to the cell body. This process affects the histone regulating enzymes in the nucleus causing more genetic activity to make needed materials for transport. Chemical signals can, also, increase transport of more messenger RNA vesicles. Special factors, like NGF, can signal production of proteins from messenger RNAs in distant sites.


Surprisingly, even before injury, vesicles filled with messenger RNA are placed along the axon, ready to be used when needed.

Transport Needs Energy

From Muessig
The transport motors stepping along the axon use ATP as their energy source. Kinesin-1 uses one ATP for every step of 8 nm. Each large vesicle or organelle has multiple motors walking. If there are conflicts and tug of wars, the amount of energy is increased. A typical transit can use millions of energy molecules. Dynein’s steps are much larger, up to 32nm, but it can go backwards and sideways as well as forward. Dynein uses many motors per vesicle—up to a dozen. This process, also uses a million energy molecules.
Given the large amount of energy required for axon transport, it is not surprising that a second mechanism has been discovered unique to transport. While mitochondria probably supply most of the energy, it appears that vesicles have glycolytic enzymes that produce energy particles linked to the vesicles as they travel on the microtubules.

In fact, the energy needs of neuronal baseline activity are very high and transport by motors is not a great percentage of the total. The brain, weighing three pounds, uses at least 20% of all the energy of the body. Much of the usage is the constant electrical activity along the axons, which requires maintenance of a large number of membrane channels. The vesicle energy backup system could work in the regions between mitochondria. This type of energy can only be used for the larger cargoes with vesicles.


Another important factor is the energy support from glia cells. Glia provide materials for glycolysis as well.

The Enormous Complexity of Transport Along the Axon

A mental event triggers instantaneous building and tearing down of scaffolding molecules. The scaffolding molecules are critical for every function of the neuron, including rapid changes of neuroplasticity. Somehow, they are the circuit boards and Lego blocks of information flow in the neuron. It is hard to imagine how this direction can occur or where it is coming from.




From R Knightley
Because of this, some scientists suspect there might be quantum computer activity in the middle of the microtubule. This information at the quantum scale would communicate with other tubules and direct the microtubules and other molecules. Such a scenario, also, fits a theory of mind interacting with quantum states.
In any case, something must direct these massively complex, shifting structures. Does a neuron think with its microtubules? How does this process connect with human thinking?
The transport microtubule system is extremely complicated, where very specific multiple motors, factors and adaptors operate together to transport many different kinds of cargoes. The complexity of the transport system includes tagging and choosing each molecule for specific cargos and specific destinations, while choosing multiple unique co factors and adaptors. Totally different assemblies are chosen for building materials, neurotrophins, mitochondria, and large vesicles like lysosomes. Each uses multiple motors at a time. It is difficult to imagine the direction for all these processes.
With all of this complex activity and regulation responding instantly to thought and the alterations of neuroplasticity, how can anyone think that this process is in any way random?


http://jonlieffmd.com/blog/are-microtubules-the-brain-of-the-neuron

Microtubules are the movers and shakers of the cell interior. 1 With the exception of the nucleus and microfilaments, virtually every organelle in the cell is placed and maintained in its functional location by the action of microtubules operating in conjunction with motor proteins. In addition, using the same motor proteins, microtubules accomplish the movement in the cell of most vesicles, including secretory vesicles, transfer vesicles, lysosomes, and endosomes. When vesicles are maintained in place in the cell, for example pigment granules, microtubules and their motors appear to coordinate with the actions of microfilaments and myosins. Coordination also functions near the plasma membrane, where microtubules do not reach. Vesicles on microtubules are transferred to microfilaments for the final micron or so to the membrane.

Microtubules come in three flavors: single, double, and triple. Single microtubules have thirteen "protofilaments", i.e. single chains of tubulin dimers, in their circumference. In cilia and flagella, double microtubules exist, with the second tubule using three protofilaments of the primary, and ten additional protofilaments. Logic suggests that these double microtubules would be more rigid than the single ones, but the ciliary structure nevertheless is constructed to be motile. Finally, triple microtubules form the structure of centrioles, as shown in the inset of the figure below.



Cross-linking between microtubules does not appear to extensively occur. They do form networks with intermediate filaments (to be described), however, and in fact the locations of intermediate filaments, in a cell like a fibroblast, overlay and depend upon those of microtubules. The links may result from globular domains of intermediate filament proteins, or cross-linking specialty proteins generally called Microtubule-Associated-Proteins (MAPs). The relationship between microtubules and intermediate filaments is particularly intimate in neuronal axons, where intermediate filaments provide tensile strength and microtubules a vesicular transport system. The cross-links between microtubules and membranous organelles, on the other hand, are the result of interactions with microtubule motor proteins called kinesins and dyneins. The same proteins are responsible for vesicular transport.

A number of other proteins bind to microtubules in order to fulfill a variety of functions. Some stabilize the tubules, some cap the tubules to prevent or to accomplish destabilization of the (+) ends of the microtubules. Microtubules are subject to catastrophic (i.e. very rapid) disaggregation beginning at the (+) end. This is thought to reflect a dynamic property of microtubules permitting them to change length and location rapidly in cells undergoing dynamic processes. Like microfilaments, the subunits of microtubules are nucleotide binding proteins, in this case GDP/GTP. Also like microfilaments, tubulin dimers add to the (+) end of microtubules in the GTP form (actually two GTPs, but one is inert). After addition, one GTP in each dimer hydrolyzes to the GDP form, which has lower affinity for the neighboring dimers, and destabilizes the microtubule. As long as GTP-tubulin dimers cap the (+) end of the microtubule it will continue to grow. But if growth slows and the end dimers have a chance to hydrolyze to the GDP forms, the microtubule will begin an explosive depolymerization from the (+) end towards the (-) end.

Microtubules are highly stabilized by MAPs in neuronal axons. During mitosis, a number of cross-linking proteins and specialized motor proteins function to connect microtubules to the mitotic spindle and to chromosomes.

Two families of proteins transport "cargo" on microtubules and localize organelles such as the Golgi apparatus and the endoplasmic reticulum. Both families of proteins have numerous members. The simplest motors structurally are the kinesins that generally, but not always, move towards the positive end of microtubules. Endoplasmic reticulum proteins have binding sites for kinesins that walk towards the (+) end of microtubules and therefore towards the plasma membrane.
The other family of motors is that of the dyneins, that generally walk towards the (-) end of microtubules. The Golgi apparatus has binding receptors for dyneins on some of its membrane proteins that push it towards the (-) ends of microtubules, and therefore it is located close to the nucleus and the centrosome, the home of the (-) ends.

There are two classes of dyneins, one is termed cytoplasmic dynein, and it is the variety that participates in vesicle movement and organelle placement. The other class is called ciliary dynein, and it, as expected, functions in cilia and flagella. This dynein is very important to the function of cilia, and loss-of-function mutations in ciliary dynein lead to a condition known as Kartagener’Syndrome. This condition typically involves severe lung disease and sterility in males.

1) http://classes.kumc.edu/som/cellbiology/cytoskeleton/microtubules/tut2.html

Live visualizations of single isolated tubulin protein self-assembly via tunneling current: effect of electromagnetic pumping during spontaneous growth of microtubule 1

As we bring tubulin protein molecules one by one into the vicinity, they self-assemble and entire event we capture live via quantum tunneling. We observe how these molecules form a linear chain and then chains self-assemble into 2D sheet, an essential for microtubule, —fundamental nano-tube in a cellular life form. Even without using GTP, or any chemical reaction, but applying particular ac signal using specially designed antenna around atomic sharp tip we could carry out the self-assembly, however, if there is no electromagnetic pumping, no self-assembly is observed. In order to verify this atomic scale observation, we have built an artificial cell-like environment with nano-scale engineering and repeated spontaneous growth of tubulin protein to its complex with and without electromagnetic signal. We used 64 combinations of plant, animal and fungi tubulins and several doping molecules used as drug, and repeatedly observed that the long reported common frequency region where protein folds mechanically and its structures vibrate electromagnetically. Under pumping, the growth process exhibits a unique organized behavior unprecedented otherwise. Thus, “common frequency point” is proposed as a tool to regulate protein complex related diseases in the future.

High frequency electromagnetic and mechanical oscillations of proteins in the literatures: A common megahertz frequency domain. High frequency electromagnetic oscillations and the effect of temperature in proteins is an exciting field of research1, because of their potential applications in the medical science. Distinct vibrational modes of proteins in the high frequency domain stems from intra-molecular degrees of freedom of proteins, every single drug molecule and biological essentials have a specific vibrational signature2. In proteins, depending on the vibrational mode triggered by an external electromagnetic frequency, the relaxation time could change from fifty nanoseconds (109 Hz ~ GHz) to a few hundred microseconds (106 Hz ~ MHz)3. However, physical protein folding, if it is very fast then takes a few microseconds and if slow then a few seconds (Hz)4. Therefore, electromagnetic oscillations are faster than microseconds, while, mechanical oscillations sustain for microseconds and higher. Obviously, for some proteins there is a time gap between electromagnetic and mechanical oscillations, for some proteins the survival for microseconds is the “common frequency point”. In this case, the common point is the inverse of microseconds that is the megahertz frequency. In summary, if in a particular protein, the electromagnetic and mechanical oscillations have a common time or frequency region where, both electromagnetic and mechanical oscillations merge, then we might manipulate one with another. Since mechanical oscillations are local, can we modulate the protein folding & its complex formation from isolated proteins using megahertz electromagnetic signals, non-locally? Here, we make a journey from atomic scale live imaging of protein complex formation to the verification of the observations made in the cell-like environment and explore the common megahertz frequency region for the tubulin protein, which is 0.8 to 8 microseconds5. It means for tubulin protein if we pump electromagnetic signal, around 2.25 MHz and 0.225 MHz, then the protein complex i.e. microtubule formation would unravel unprecedented features.



1) http://www.nature.com/articles/srep07303

Regulation of microtubule nucleation by Ran protein signal transduction

https://reasonandscience.catsboard.com/t2096-the-astonishing-language-written-on-microtubules-amazing-evidence-of-design#7189

Microtubules are polarised polymers involved in a wide range of cellular processes including chromosome separation, intracellular transport, organelle positioning, cell–cell signalling and cell motility 2 Tight spatial and temporal regulation of the formation, organisation, and dynamic behaviour of the microtubule cytoskeleton is extremely important. Consequently, cells have  complex mechanisms to regulate microtubule nucleation, polymerisation and catastrophe, severing, stabilisation and transportation. Collectively, these processes establish diverse microtubule networks with highly specialised functions in different cells or at different times during the cell cycle.





Any series of molecular signals in which a Ran GTPase relays one or more of the signals resulting in the modulation of the rate, frequency or extent of microtubule nucleation. 1



Regulation of microtubule nucleation by Ran protein signal transduction

https://reasonandscience.catsboard.com/t2096-the-astonishing-language-written-on-microtubules-amazing-evidence-of-design#7189

Microtubules are polarised polymers involved in a wide range of cellular processes including chromosome separation, intracellular transport, organelle positioning, cell–cell signalling and cell motility 2 Tight spatial and temporal regulation of the formation, organisation, and dynamic behaviour of the microtubule cytoskeleton is extremely important. Consequently, cells have complex mechanisms to regulate microtubule nucleation, polymerisation and catastrophe, severing, stabilisation and transportation. Collectively, these processes establish diverse microtubule networks with highly specialised functions in different cells or at different times during the cell cycle.

Evolution ? Hmm ?? Regulation, separation, positioning, stabilisation are all terms usually attributed to the action of intelligence. Either the tight regulation of microtubule regulation was right from the beginning, or no deal. This is extraordinary evidence of design.

Signalling requires the establishing of a signalling code, and that code has to be used to preprogramm information, and signalling networks and channels are required in order to establish communication, which goes from the sender, through the network, to the receiver which can interpret and understand the signal and react accordingly. This is overwhelming evidence of intelligent setup, which is not rationally explained by gradative evolution. It is an all or nothing business.

1. https://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0090226
2. https://portlandpress.com/essaysbiochem/article/62/6/765/78500/Microtubule-nucleation-by-tubulin-complexes-and

Neurons = sophisticated computers, made of transistors, and memristors

https://reasonandscience.catsboard.com/t2096-the-astonishing-language-written-on-microtubules-amazing-evidence-of-design#9070

Intelligent Design should predict, that the more science advances, the more levels and layers of complexity should be unraveled on a sub-cellular level. The more complex, the more that should indicate design:

What does complex mean?
https://reasonandscience.catsboard.com/t3112-what-does-complex-mean

I knew that neurons are transistors:
https://reasonandscience.catsboard.com/t2292-neurons-remarkable-evidence-of-design#7201

and now science is also unraveling, that neurons are also memristors, and scientists are adopting the technology for human computing devices:

RESEARCHERS UNVEIL ELECTRONICS THAT MIMIC THE HUMAN BRAIN IN EFFICIENT, BIOLOGICAL LEARNING 2
Only 10 years ago, scientists working on what they hoped would open a new frontier of neuromorphic computing could only dream of a device using miniature tools called memristors that would function/operate like real brain synapses. But now a team at the University of Massachusetts Amherst has discovered, while on their way to better understanding protein nanowires, how to use these biological, electricity conducting filaments to make a neuromorphic memristor, or “memory transistor,” device. It runs extremely efficiently on very low power, as brains do, to carry signals between neurons.

Jack A.Tuszynski Microtubules as Sub-Cellular Memristors 2020 3

Memristors represent the fourth electrical circuit element complementing resistors, capacitors, and inductors.
https://en.wikipedia.org/wiki/Resistive_random-access_memory

Hallmarks of memristive behavior include pinched and frequency-dependent I–V hysteresis loops and most importantly a functional dependence of the magnetic flux passing through an ideal memristor on its electrical charge. Microtubules (MTs), cylindrical protein polymers composed of tubulin dimers are key components of the cytoskeleton. They have been shown to increase solution’s ionic conductance and re-orient in the presence of electric fields. It has been hypothesized that MTs also possess intrinsic capacitive and inductive properties, leading to transistor-like behavior. Here, we show a theoretical basis and experimental support for the assertion that MTs under specific circumstances behave consistently with the definition of a memristor. Their biophysical properties lead to pinched hysteretic current-voltage dependence as well a classic dependence of magnetic flux on electric charge. Based on the information about the structure of MTs we provide an estimate of their memristance. We discuss its significance for biology, especially neuroscience, and its potential for nanotechnology applications.

l our findings in the experiments and simulations seem to indicate that MTs can propagate, and amplify, electric signals via ionic flows along the MT surface, through the lumen, and across their nanopores. Signals carried by ionic flows along MTs can be incorporated into the functions of neurons, and can affect ion channels, for example. Our discovery that MTs are biological memristors could, for example, help resolve the heretofore unknown  origin of the impressive memory capabilities exhibited by the amoeba, which do not have neurons, let alone a  brain, but are loaded with MTs.

The first physical realization of a memristor was achieved in 2008 and it has held a promise of nanoelectronics beyond Moore’s law, although this realization has been both difficult and controversial.

The ever-growing need of computing power to handle data intensive applications is seriously challenging conventional digital von Neumann computing architectures. The physical separation between the computing and memory units can indeed generate long latency time and large energy consumption when data intensive tasks are performed. In this context, researchers look at the emerging nanoscale analog devices, such as memristors, as a viable approach for developing new computing paradigms, based on in-memory and analog computation, which are potentially capable to overcome the limitations of conventional computer architectures . The memristor (a shorthand for memory resistor) is the fourth basic passive circuit element theoretically introduced by Prof. Leon Chua in 1971. The appealing features of a memristor are non-volatility, i.e., the memristor capability to hold data in memory without the need of a power supply, and non-linearity, which makes memristor circuits capable to generate quite a rich variety of oscillatory and complex dynamics. The combination of these two features enables in-memory computing, i.e., the co-location of computation and memory in the same device

In-memory computing, which resembles a basic principle of brain computation, can provide several advantages, such as low energy consumption, high bandwidths, excellent scalability, thus lending itself as quite a promising novel computing approach in the field of artificial intelligence and big data.

My comment:  As this demonstrates, the best, most sophisticated computer hardware technology is implemented in our brain, in neurons. A technology, that we humans, are only now starting to unravel, understand, and implement in our human-made computers. if that is not evidence of intelligent design, i don't know, what is.....

1. https://www.frontiersin.org/articles/10.3389/fnins.2021.681035/full
2. https://www.umass.edu/news/article/researchers-unveil-electronics-mimic-human
3. https://www.nature.com/articles/s41598-020-58820-y