Eyes, eyespots, Volvox ,and interdependence

Origin of eyespots - supposedly one of the simplest eyes

https://reasonandscience.catsboard.com/t2638-volvox-eyespots-and-interdependence#5768

Rhodopsins falsify the claim of evolution of the eye

https://reasonandscience.catsboard.com/t2638-eyes-eyespots-volvox-and-interdependence#6573

Channelrhodopsins are the first and most essential and important players of vision. They are expressed in a specialized compartment - the so-called eyespot- where light initiates a fast inward-directed photocurrent. The electrical signal is amplified by the activation of voltage-gated secondary channels and is transmitted to the two flagella which in turn adjust their beating plane, frequency and pattern. Hence, the complex interplay of photoreceptors and flagella movement enables the algae to perform positive or negative phototaxis according to the quality of the ambient light.
Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. embed in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein.  

Channelrhodopsin has only function by the interplay conjoined with retinal, and as such, they are interdependent. 

Evolution of Rhodopsins
Opsins are a group of proteins that underlie the molecular basis of various light sensing systems including phototaxis, circadian (daily) rhythms, eyesight, and a type of photosynthesis. Opsins are retinal proteins because they bind to a light-activated, non-protein chromophore called retinal. All opsin proteins are embedded in cell membranes, crossing the membrane seven times.  Functional residues, such as those within the catalytic sites of enzymes, are highly constrained and thus well conserved across organisms because mutations within these sites are normally deleterious. 

That raises the question of how these precursors of opsins,  G Proteins, emerged in the first place since they are highly specific and prone to mutations. 

An often cited source of evolutionary novelty is the recruiting and co-option of extant building blocks, and incorporate them into new systems, by natural selection of new functions. Rhodopsin would have to undergo evolution by recruiting All-trans-retinal chromophores, which it would have to find ready fully formed and functional, and finely tuned and right-sized to fit the binding pocket. Retinal is  a molecule obtained by a complex multistep biosynthesis pathway starting with carotenoid chromophores from fruits, flowers, trees or vegetables.  It would require elaborated import mechanisms and the information how to be inserted it in the binding pocket, and attached at the right place, and the insertion of a protonated retinal Schiff base ( The term Schiff base is normally applied to these compounds when they are being used as ligands ) 

The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. 

The binding of the chromophore to the opsin is essential to trigger the conformational change which in sequence triggers the signal transduction pathway, which transmits a signal to the brain, ion channels for flagella beating etc. and must be precise and functional from the beginning. Following is required :  

1. a Schiff base linkage
2. a Lys296 residue where chromophore retinal covalently binds
3. the side chain of the residue
4. an essential amino acid residue called "counter ion". The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. 17
5. There is a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of  rhodopsin

Unless all of these specific points were not just right from the beginning, rhodopsin would not be functional. Each of these processes demands already coordinated and finetuned interplay and precise orchestration between opsin and retinal. 

We read in the book: Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105
Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if subsystems or parts are put together in the right order, they also need to interface correctly.

The first question is: How did opsins and their configuration of seven precisely folded alpha helix transmembranes emerge, and later, the precise binding sites for retinal, our second essential player in sight? 

Rao et. al. have proposed that "...the packing of seven helices together may represent a uniquely stable arrangement that has been achieved through a process of convergent evolution." 

Here we go. We " have proposed ".... convergent evolution. But but.... where is the evidence ??

In the paper: The Origins of Novel Protein Interactions during Animal Opsin Evolution, the authors make the remarkable admittance: 

Genetic changes are known to modify phenotype during evolution by altering the interactions between a protein and its ecological or biochemical environment, by modulating existing protein-protein interaction. However, the specific genetic changes that give rise to the evolutionary origins of novel protein-protein interactions HAVE RARELY BEEN DOCUMENTED IN DETAIL.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001054

The origin of correct protein folding is a major problem in evolutionary biology. The precision upon which opsins must fold into their seven transmembrane configurations is staggering: 

Biophysicists at JILA have measured protein folding in more detail than ever before, revealing behavior that is surprisingly more complex than previously known. . . .2 the JILA team identified 14 intermediate states—seven times as many as previously observed—in just one part of bacteriorhodopsin, a protein in microbes that converts light to chemical energy and is widely studied in research. “The increased complexity was stunning,” said project leader Tom Perkins, a National Institute of Standards and Technology (NIST) “Better instruments revealed all sorts of hidden dynamics that were obscured over the last 17 years when using conventional technology.” “If you miss most of the intermediate states, then you don’t really understand the system,” he said. Knowledge of protein folding is important because proteins must assume the correct 3-D structure to function properly. Misfolding may inactivate a protein or make it toxic. Several neurodegenerative and other diseases are attributed to incorrect folding of certain proteins. 
https://www.nist.gov/news-events/news/2017/03/jila-team-discovers-many-new-twists-protein-folding

An article in Nature magazine confirms :
Even as far back as the prokaryotes the complex seven transmembrane domain arrangement of opsin molecules seems to prevail without simpler photoreceptors existing concurrently. Darwin’s original puzzle over ocular evolution seems still to be with us but now at a molecular level. 
https://www.nature.com/articles/eye2015220

This is a KEY-confession which basically puts to an end all claims that the evolution of the eye is a solved problem, and that the evidence has been found. 
 
Retinal 
Channelrhodopsins are directly light-gated channels that contain retinal, which undergoes 13-trans to cis isomerization upon illumination. Is a unique molecule with a chemical design that allows optimal interaction with the opsin protein in its binding pocket, and this is essential for the formation of the light-activated conformation of the receptor. When a photon strikes this retinal chromophore and the light energy is absorbed by retinal, this light energy is used to cause one of the alkenes in retinal to undergo a  configuration change. This configuration change causes a change in the conformation (the three-dimensional shape) of the opsin protein , which triggers the complex transduction cascade.  All structural details in the retinal chromophore are functionally important. 

A paper reports an intriguing evolutionary conservation of the key components involved in chromophore production and recycling. The synthesis of retinal precedes a complex pathway of several enzymatic steps starting from carotenoids molecules. There would have been no evolutionary advantage to evolve such a pathway and its proteins, unless there was the know how to make the molecule with the correct structure, in order to work fine and fit correctly in the opsin pocket to form a functional Rhodopsin protein. 










Nilsson's famous paper on eye evolution starts with an eyespot, and in a nice row of pictures shows how eyespots could have evolved to complex camera eyes :



Representative stages of a model sequence of eye evolution.
In the initial stage (1) the structure is a flat patch of light-sensitive cells sandwiched between a transparent protective layer and a layer of dark pigment 1

In the last sentence of the paper, which has been used since it was published in 1994 as a reference to back up the claim of the evolution of  eyes, Nilsson writes :

" the eye was never a real threat to Darwin's theory of evolution."

In the article  Light and the evolution of vision in Nature magazine, the author writes: 
Chlamydomonas is green algae in the plant kingdom. Phototaxis is essential for it; moving towards light upon which they depend for energy and nutrition, yet also undergoing negative phototaxis to protect themselves against too intense a source of illumination. The eyespot is not the photoreceptor itself but rather a mass of carotenoid pigment shading the photoreceptor from light from one direction. This demonstrates the essential components of any visual system; any photosensitive organism needs a photoreceptor that detects the light. But that alone would not allow the organism to determine the direction of the light source. A pigment spot reduces the illumination from one direction, or changes the wavelength of the incident light falling on the photoreceptor, thus allowing the organism to move in the direction of the light or away for it. So third, a mechanism to promote movement is essential. To detect the light is one thing but to move towards or away from it requires a motor system; the flagellae in Chlamydomona. But also a mechanism is required by which detection of light can be translated into a change in flagellar movement, generally an ion flux of one kind or another.

This demonstrates the essential components of this visual system:

1. any photosensitive organism needs a photoreceptor that detects the light. 
2 A pigment spot reduces the illumination from one direction, or changes the wavelength of the incident light falling on the photoreceptor, thus allowing the organism to move in the direction of the light or away for it.
3 a mechanism to promote movement is essential. it requires a motor system; the flagellae
4- But also a mechanism is required by which detection of light can be translated into a change in flagellar movement, generally an ion flux of one kind or another. Trans-membrane calcium flux initiates a cascade of electrical responses causing depolarization of the cell and ultimately controls the flagellar beating pattern.




This is an interdependent system composed of 4 essential components, photoreceptors, a pigment spot, the flagellae, and ion flux, of which, if one is missing, the organism cannot move by phototaxis. Natural selection would not select any intermediate evolutionary step, since the system, with any of the four elements missing, would confer no function, and no advantage of survival. 

From the kinetics and the rhodopsin action spectrum of these photocurrents we conclude that they are part of the rhodopsin-regulated signal transduction chain controlling the cellular behaviour in light. Both photocurrents are Ca²⁺-dependent and are suppressed by the Ca²⁺-channel inhibitors verapamil and pimozide, suggesting that the photoreceptor current and probably the flagellar current are both carried by Ca²⁺. 5




Question: Why did the authors begin their narrative with a " flat patch of light-sensitive cells "? rather than  with an explanation of how such a "patch" could have evolved? and what it would be good for, unless being there for a specific function, like vision, detection of light/shading, circadian rhythm etc., which always requires other parts that constitute a system of at least two parts? 

The claim is that a unicellular, "simple" organism, like Volvox green algae, may have been equipped with a photoreceptor organelle, and the eyes may have evolved from such an ancestral state. The story of proponents of the evolution of the eye goes as follows: The simple light-sensitive spot on the skin of some ancestral creature gave it some tiny survival advantage, perhaps allowing it to evade a predator. 1

There is IMHO nothing " simple ": eyespots like in Chlamydomonas reinhardtii algae, have 742 different proteins 8 ; they have an elaborate structure, and a  high ultrastructural complexity 18  .     
Zoologist Dan-Erik Nilsson avoided asking the relevant question: What good is an eyespot for by itself? 

From the "simplest", most rudimentary eye forms, like eyespots,  to complex vertebrate eyes, like our camera eyes, rhodopsins are the first players in a complex chain of biochemical events. In unicellular organisms, like Chlamydomonas, eyespots shade dark from the light and interconnected with the flagellum, they either distance from clarity, or move closer to sunlight, depending on their needs. This is an interdependent system, where one has no function unless linked to the other.

Rhodopsin is the central player in vision. There is no vision without it. Unless rhodopsin transforms light into a signal, and that signal is used by a signal transduction pathway to promote phototaxis, neither rhodopsins nor eyespots would bear function by their own. A flagellum cannot rotate to move the cell in the right direction unless it gets the right instructions.

The design of the eyespot apparatus in conjunction with the helical movement of the cell produces a highly directional optical device allowing effective tracking of the light direction. In Chlamydomonas reinhardtii, the eyespot apparatus is usually composed of two layers of highly ordered carotenoid-rich lipid globuli that are situated at the periphery of the chloroplast. The globuli layers are subtended by thylakoid membranes. Additionally, the outermost globule layer is attached to specialized areas of the chloroplast envelope membranes and the adjacent plasma membrane.

The carotenoid layers reflect a light beam and amplify the light signal from the outside of the cell on rhodopsin (the “front side”) and block the light from the inside of the cell (the “rear side”).  These carotenoid granules are crucial for phototaxis. Crucial = indispensable - irreducible. As the cell swims with self-rotation, the eyespot apparatus scans the incident light around the cell’s swimming path. After photoreception by the rhodopsins, the cell changes the beating balance of the two flagella and exhibits either positive or negative phototaxis (swimming toward or away from the light source, respectively).

The phototactic pathway primarily consists of four steps: 

(i) photoreception by Channel rhodopsins
(ii) excitation of the cellular membrane; 
(iii) increase in intraflagellar [Ca2+]; and 
(iv) a change in the beating balance between the two flagella, i.e., the cis-flagellum (the one closest to the eyespot) and the trans-flagellum (the one farthest from the eyespot)


Important cellular processes result from the concerted action of multiple proteins organized in complex networks. Studies in evolution have revealed how individual proteins can acquire new functions due to changes in their binding specificity or catalytic potential. However, these characteristics alone often cannot explain the evolution of complex cellular functions, because network output does not solely depend on the function of an individual protein, but rather on the integrated function of multiple components with intricate regulatory relationships. 2

This is what we observe even in unicellular organisms like Chlamydomonas where motility depends on a multitude of different proteins interconnected and highly regulated to adapt to the different environmental conditions.

Rhodopsin Structure and Activation
Channelrhodopsins, the first players of vision,  are expressed in a specialized compartment - the so-called eyespot- where light initiates a fast inward-directed photocurrent. The electrical signal is amplified by the activation of voltage-gated secondary channels and is transmitted to the two flagella which in turn adjust their beating plane, frequency and pattern. Hence, the complex interplay of photoreceptors and flagella movement enables the algae to perform positive or negative phototaxis according to the quality of the ambient light. 1

Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. embed in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein. 3  

Channelrhodopsin has only function conjoined with retinal.

Evolution of Rhodopsins
Opsins are a group of proteins that underlie the molecular basis of various light sensing systems including phototaxis, circadian (daily) rhythms, eye sight, and a type of photosynthesis. Opsins are  retinal proteins because they bind to a light-activated, non-protein chromophore called retinal. All opsin proteins are embedded in cell membranes, crossing the membrane seven times. 6

Functional residues, such as those within the catalytic sites of enzymes, are highly constrained and thus well conserved across organisms because mutations within these sites are normally deleterious. 3

That raises the question how these G Proteins emerged in the first place since they are highly specific and prone to mutations.

An often cited source of evolutionary novelty is the recruiting and co-option of extant building blocks, and incorporate them into new systems, by natural selection of new functions. Rhodopsin would have to undergo evolution by recruiting All-trans-retinal chromophores, which it would have to find ready fully formed and functional, and finely tuned and right-sized to fit the binding pocket,  a molecule obtained by a complex multistep biosynthesis pathway starting with carotenoid chromophores from fruits, flowers, trees or vegetables. 4  It would require elaborated import mechanisms and the information how to insert it in the binding pocket, and attached at the right place, and the insertion of a protonated retinal Schiff base ( The term Schiff base is normally applied to these compounds when they are being used as ligands )

The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. 14

The binding of the chromophore to the opsin is essential to trigger the conformational change and must be precise and functional from the beginning. Following is required : 

1. a Schiff base linkage
2. a Lys296 residue where chromophore retinal covalently binds
3. the side chain of the residue
4. an essential amino acid residue called "counter ion". The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. 17
5. There is a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of  rhodopsin

Unless all of these specific points were not just right from the beginning, rhodopsin would not be functional. Each of these processes demands already coordinated and finetuned interplay and precise orchestration between opsin and retinal.

Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105

Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if subsystems or parts are put together in the right order, they also need to interface correctly.


The question is: How did opsins and their configuration of seven precisely folded alpha helix transmembranes emerge?

Rao et. al. have proposed that "...the packing of seven helices together may represent a uniquely stable arrangement that has been achieved through a process of convergent evolution." 10

Here we go. We " have proposed ".... convergent evolution. But but.... where is the evidence ??

In the paper: The Origins of Novel Protein Interactions during Animal Opsin Evolution, the authors make the remarkable admittance: 

Genetic changes are known to modify phenotype during evolution by altering the interactions between a protein and its ecological or biochemical environment, by modulating existing protein-protein interaction. However, the specific genetic changes that give rise to the evolutionary origins of novel protein-protein interactions HAVE RARELY BEEN DOCUMENTED IN DETAIL.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001054

Origin of correct protein folding, a major problem in evolutionary biology

The precision upon which opsins must fold into their seven transmembrane configuration is staggering:

Biophysicists at JILA have measured protein folding in more detail than ever before, revealing behavior that is surprisingly more complex than previously known. . . .2 the JILA team identified 14 intermediate states—seven times as many as previously observed—in just one part of bacteriorhodopsin, a protein in microbes that converts light to chemical energy and is widely studied in research. “The increased complexity was stunning,” said project leader Tom Perkins, a National Institute of Standards and Technology (NIST) “Better instruments revealed all sorts of hidden dynamics that were obscured over the last 17 years when using conventional technology.” “If you miss most of the intermediate states, then you don’t really understand the system,” he said. Knowledge of protein folding is important because proteins must assume the correct 3-D structure to function properly. Misfolding may inactivate a protein or make it toxic. Several neurodegenerative and other diseases are attributed to incorrect folding of certain proteins.
https://www.nist.gov/news-events/news/2017/03/jila-team-discovers-many-new-twists-protein-folding

An article in Nature magazine confirms :
even as far back as the prokaryotes the complex seven transmembrane domain arrangement of opsin molecules seems to prevail without simpler photoreceptors existing concurrently. Darwin’s original puzzle over ocular evolution seems still to be with us but now at a molecular level. 4
https://www.nature.com/articles/eye2015220



Retinal chromophores: 
channelrhodopsin-1 and channelrhodopsin-2 (ChR-1 and ChR-2), are directly light-gated cation channels that contain a planar all-trans, 6-S-trans retinal chromophore, which undergoes 13-trans to cis isomerization upon illumination.

Retinal is a unique molecule with a chemical design that allows optimal interaction with the opsin apoprotein in its binding pocket, and this is essential for the formation of the light-activated conformation of the receptor. 2  When a photon strikes this retinal chromophore and the light energy is absorbed by retinal, this light energy is used to cause one of the alkenes in retinal to undergo a  configuration change. This configuration change causes a change in the conformation (the three-dimensional shape) of the opsin protein , which triggers the complex transduction cascade. 



All structural details in the retinal chromophore are functionally important

A paper reports an intriguing evolutionary conservation of the key components involved in chromophore production and recycling. The synthesis of retinal precedes a complex pathway of several enzymatic steps starting from carotenoids molecules. There would have been no evolutionary advantage to evolve such a pathway and its proteins, unless there was the know how to make the molecule with the correct structure, in order to work fine and fit correctly in the opsin pocket to form a functional Rhodopsin protein.


Carotenoids biosynthesis in Chlamydomonas reinhardtii





Schematic diagram of the carotenoid biosynthetic pathway in plants and microalgae.

Phytoene synthase (PSY) catalyses the first step in the carotenoid specific pathway, which leads the carbon flux towards carotenes and xantophylls production.
IPP isopentenyl pyrophosphate,
DMAPP dimethylallyl pyrophosphate,
GGPP geranylgeranyl pyrophosphate,
GGPPS geranylgeranyl pyrophosphate synthase,
PDS phytoene desaturase,
Z-ISO 15-cis-ζ-carotene isomerase,
ZDS ζ-carotene desaturase, CRTISO carotene isomerase,
LCYb lycopene β-cyclase,
LCYe lycopene ε-cyclase,
P450b-CHY cytochrome P450 β-hydroxylase,
P450e-CHY cytochrome P450 ε-hydroxylase,
CHYb carotene β-hydroxylase,
BKT β-carotene oxygenase,
ZEP zeaxanthin epoxidase,
violaxanthin de-epoxidase

Retinal, the chromophore that is covalently linked to the rhodopsin-type photoreceptors of the eyespot apparatus likely results from symmetric cleavage of β-carotene by a β -carotene-15,15  -oxygenase (BCO). candidate genes related to the animal enzyme have been identified in Chlamydomonas

We have by this only scratched the surface. We would have to explain the precise interplay and complex mechanism between the eyespot and the flagella, describe the flagellum in all its complexity. But what has been demonstrated so far is, that eyespots are FAR FROM simple, and depend on many interdependent parts, which, if not fully interconnected and regulated, would not permit Volvox to swim either towards the light (positive phototaxis) or away from the light. 

We can safely say: Vision and its origin is best explained by intelligent design

The eyespot plays an accessory role in photobehavioral responses and eyeless mutant would be able to perceive and respond to light. Recent study using the reactive oxygen species (ROS), leads to the identification of novel eyeless mutant of Chlamydomonas exhibiting strong phototaxis responses. These reports support the earlier made hypothesis, that the photoperception in Chlamydomonas is not confined to eyespot. One possibility is that the bacterial rhodopsins localized in the flagella of Chlamydomonas might be responsible for a non-directional phototransduction of this organism. The eyespot-guided phototaxis is very important for the zooplankton larvae of marine invertebrates and is proposed to mediate larval swimming towards the light. Recently, it has been proposed that eyespot localized opsin of the Platynereis are the photoreceptors for controlling phototaxis of this organism. It would be interesting to elucidate the role of intraflagellar transport (IFT) machinery in the trafficking of opsin in the eyespot of the Platynereis, which would shed light on evolutionary link of the of the IFT mediated trafficking of the rhodopsin(s) in nature. The involvement of IFT in the eyespot localization of rhodopsin would further support its role in intracellular trafficking of proteins similar to the case of immune synapse assembly in higher animals.

1. http://faculty.jsd.claremont.edu/dmcfarlane/bio145mcfarlane/PDFs/Nilsson%20and%20Pelger_eye%20evolution%20model%20ProcRoyalSoc_1994.pdf.
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976203/
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177186/
4. http://www.nature.com/eye/journal/v30/n2/full/eye2015220a.html
5. https://sci-hub.tw/https://www.nature.com/nature/journal/v351/n6326/abs/351489a0.html

Volvox , eyespots, and interdependence

https://reasonandscience.catsboard.com/t2638-volvox-eyespots-and-interdependence

The volvocine algae include both unicellular and multicellular organisms that are closely related and exist today (Kirk 1998). The unicellular species in this group is named Chlamydomonas reinhardtii (hereafter Chlamydomonas), and its best-studied, close multicellular relative is a species named Volvox carteri (hereafter Volvox). 10

Researchers believe that the last common ancestor of the present-day volvocine algae was a unicellular species closely resembling modern-day Chlamydomonas and that Chlamydomonas may not have changed much at the genetic level, with respect to that ancestor.

The origin of photoreceptor cells is largely a matter of speculation. Even though bacteria have sensory rhodopsins there is no detectable sequence homology between bacteriorhodopsins and metazoan visual rhodopsins . However, the two may still be phylogenetically related, since their three-dimensional structures with seven membrane-embedded _-helices are very similar, and they all share the same chromophore, retinal, which is attached in a Schiff base linkage to a lysine residue in the seventh _-helix. The most primitive organism in which a rhodopsin has been found which may be related to animal opsins is Volvox, a colonial green alga in the transition zone between protists and multicellular organism.  Photoreception probably initially evolved in cyanobacteria which later were taken up as symbionts into eukaryotic cells and gave rise to chloroplasts. During the transition from unicellular to multicellular organisms, each cell of the primitive multicellular organism, like Volvox, may have been equipped with a photoreceptor organelle, and the eyes may have evolved from such an ancestral state by cell differentiation and organogenesis.

Detecting light is arguably one of the most coveted abilities in the living world. Eyes, or their anatomical equivalents, have independently evolved many times among animals, and the consistency of their organization, featuring a cornea or lens that focuses photons onto a sensory surface, is often brought to the fore as an example of convergent evolution. 2

Very likely, opsins started with the huge advantage of being able to couple to established transduction pathways via a G-protein. The ancestral opsin appears to have been allied with a melatonin receptor or similar GPCR, as opsins form a sister group to such GPCRs in the genome of the placozoan Trichoplax adhaerens

Algal Sensory Photoreceptors 7
Because algae lack a brain, the photoreceptor system must be directly connected to an output system that modulates the swimming direction according to the deviation from the desired tracking direction. Light excitation of a Chlorophycean alga results in a cascade of electrical events. The unicellular alga Chlamydomonas possesses a visual system which guides it to places that are optimal for photosynthetic growth. The rhodopsin, serving as the photoreceptor, conveys light information into a cellular signal. This signal is transmitted via several electrical steps to the flagella, where it modulates the flagellar beating pattern.

The eyespot acts as a quarter-wave plate that reflects and intensifies light of a specific spectral range. The carotenoid layers reflect incident light back to the actual photoreceptor in the plasma membrane, and shield it from light passing through the cell body which are collectively known as the stroma, contain soluble enzymes as well as chloroplast ribosomes. 

Channelrhodopsins: light-activated ion channels
The multicellular green alga Volvox carteri uses channelrhodopsin-1 (VChR1) and VChR2
Microbial rhodopsins are photoactive, seven-transmembrane (7TM) helix receptors and use retinal as a chromophore

 
Photon absorption triggers isomerization of the retinal from the all-trans configuration to the 13-cis form (Figure 2b). Subsequently, major structural changes of the protein backbone occur and a "preopen" state is formed on a sub-nanosecond timescale. Similar to proton-pumping rhodopsins, the retinal Schiff-base transiently deprotonates, thereby forming a blue-shifted photocycle intermediate (P390).

Eyespots are the simplest and most common "eyes" found in nature, composed of photoreceptors and areas of bright orange-red pigment granules 4  Most flagellate green algae exhibiting phototaxis posses a singular specialized light sensitive organelle, the eyespot apparatus (EA). Its design principles are similar in all green algae and produce, in conjunction with the movement pattern of the cell, a highly directional optical device. It enables an oriented movement response with respect to the direction and intensity of light.  It utilizes specialized microbial-type rhodopsins, which act as directly light-gated ion channels. They use elaborate structures and the presence of retinal-based photoreceptors. 6

Functional eyespots are often ultrastructurally complex and involve usually local specializations from different compartments. The term eyespot apparatus (EA) has therefore been early introduced to differentiate between the
whole light receiving and modulating organelle and the eyespot sensu stricto (i.e. the plate(s) of carotenoid-rich lipid globules which modulate the light signal; Unicellular algae belong to the group of smallest organisms possessing primordial visual systems with a rather high degree of complexity. Their ultrastructure is diverse and employs different optical principles to monitor the light direction. Most EAs, however, act as combined screening and reflecting devices, which, in combination with the specific movement patterns of the cell, produce optimal contrast enhancement at the presumptive photoreceptor localization by speciWc absorption and back reflection of light. Thereby, highly directional photoreceptive organelles of surprising complexity are generated. 

Some Dinophyceae possess even more complex systems, the eyelike ocelloids with a lens.   Due to the elaborate structures of the EAs and the presence of rhodopsins in some algal lineages including cyanobacteria and algae are thought to play an important role in the evolution of photoreception and eyes. Following its perception by the algal photoreceptors, the light signal is converted into an electrical or chemical signal and transmitted to the flagella. This signal carries information about the relative orientation of the cell to the light direction and its intensity and quality. 

In green algae a cascade of electrical events is initiated by excitation of the channelrhodopsins, whereas in Euglena the first step is an increase in cAMP after stimulation of photoactivated adenylate cylase. Finally, the flagella mediate the corresponding movement responses in a complex stimulus and species dependent manner, since different swimming behaviours occur even within one algal lineage.

More recently, proteomic analysis of the eyespot and associated proteins reveal complex signal transduction machinery in the algal “ eye ” which contains  200 different proteins ( Schmidt et al. , 2006 ). We developed a procedure to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the identification of 202 different proteins 8
The photoreceptors identified so far are generally believed to be located in this plasma membrane patch. Phototaxis requires the cell to determine the direction of incident light. C. reinhardtii most likely accomplishes this by monitoring the modulation of the light intensity reaching its photoreceptors as the cell rolls around its longitudinal cell axis during helical forward swimming. The eyespot globule layers are important for modulation of the light intensity. They confer increased directionality and contrast to the photoreceptors by a dual action. First, they shield them from light passing through the cell body. Second, they reflect light falling directly on the eyespot that is not absorbed by the photoreceptors back onto the overlying plasma membrane. Thus, reflection amplifies the light signal at the photoreceptor location and thereby increases their excitation probability Both absorption and reflection increase the front-to-back contrast at the location of the photoreceptors up to eightfold

The signal received by the eyespot apparatus is low and nearly constant when the swimming direction of the cells is well aligned with the light direction but changes when swimming direction deviates from light direction. This periodic signal is then processed in an as yet unknown way and finally initiates corrective flagellar responses to realign the swimming path. Thus, the whole complex (i.e., the specialized membranes and the eyespot globules forming the functional eyespot apparatus) is important for optimal performance of this primitive visual system. Due to the elaborate structures of algal eyespot apparatuses and the known presence of rhodopsins in some lineages, algae are thought to play an important role in the evolution of photoreception and eyes

Volvox never stops swimming. It can respond to a change in light by turning quickly; the cells with eyespots nearest the light shut off their propellers and the active propellers cause the Volvox to turn toward the light.

All of this activity requires a complex system of biochemical communication between each eyespot and the flagella propellers. Although Volvox is supposed to be a simple creature, this complex biochemistry and cell-to-cell communication still mystifies scientists. 9

Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii 12
The eyespot contains large amounts of carotenoids and is crucial for phototaxis.  The phototactic response is triggered by photoreception by an elaborate subcellular organelle, the eyespot (Fig. 1). This organelle is observed as an orange spot located near the cell equator. It contains the carotenoid-rich granule layers in the chloroplast and the channelrhodopsin photoreceptor proteins ChR1 and ChR2 in the plasma membrane. The carotenoid layers of the eyespot function as a light reflector.

Recent studies suggested that the Chlamydomonas phototactic pathway primarily consists of four steps: 

(i) photoreception by ChRs; 
(ii) excitation of the cellular membrane; 
(iii) increase in intraflagellar [Ca2+]; and 
(iv) a change in the beating balance between the two flagella, i.e., the cis-flagellum (the one closest to the eyespot) and the trans-flagellum (the one farthest from the eyespot)

Channelrhodopsin-1 Initiates Phototaxis and Photophobic Responses in Chlamydomonas by Immediate Light-Induced Depolarization[W] 13
Rhodopsins are membrane-spanning proteins (opsins) with a covalently bound retinal serving as the chromophore. Three rhodopsin classes are distinguished by their different functions: 

(1) sensory rhodopsins operate via an enzymatic signaling system as visual photoreceptors in animal eyes or sensors for phototaxis in prokaryotes; 
(2) light-driven ion pumps for H+ and Cl− form a primordial mechanism for photosynthetic energy conversion in archaea and eubacteria
(3) channelrhodopsins mediate light-induced conductance of H+ and other cations in algal eyes

CHR1 has been reported to be selective for H+, whereas CHR2 was shown to conduct Na+, K+, and Ca2+. CHRs are thought to be the photoreceptors that, in vivo, mediate the photoreceptor current IP in the eye of Chlamydomonas and related algae. The photoreceptor current triggers phototactic and photophobic responses. The photophobic response comprises backward swimming for half a second upon sudden changes in light intensity, whereas phototaxis is the sum of biased directional changes that appear as smooth swimming toward or away from a light source at high light intensities

14


Cryo-FIB of plunge-frozen Chlamydomonas cells.
(A) Cross-section diagram of a Chlamydomonas cell. The chloroplast is shown in color. Labels: (1) flagellum, (2) cell wall, (3) eyespot, (4) nucleus, (5) plastoglobule, (6) thylakoids, (7) chloroplast envelope, ( starch granule, (9) pyrenoid, (10) chloroplast stroma, (11) plasma membrane. 
(B–E) Preparation of FIB lamellas in the dual-beam microscope. (B) Top-view scanning electron microscopy (SEM) image of Chlamydomonas cells frozen onto the holey carbon of a 200 mesh grid. 
(C) The same cells imaged with the FIB (secondary electron detection) at the milling angle of 9°. (D) Schematic of a finished lamella. The direction of FIB milling is from left to right. (E) SEM top-view of the lamella milled from the cells shown in B and C. The direction of FIB milling is from right to left. (F) TEM high-defocus montaged overview of the same lamella. Unbinned pixel size: 30.1 Å. (G) Slice from a tomographic volume acquired at the position indicated by the yellow box in F. From left to right, RuBisCO complexes of the pyrenoid (py) are surrounded by a starch sheath (ss), followed by thylakoids (th), a starch granule (sg), and the chloroplast envelope (ce). Outside of the chloroplast, Golgi stacks and rough endoplasmic reticulum can be seen. Both cytoplasmic and chloroplast ribosomes are also visible. The tomogram was 2× binned. Unbinned pixel size: 9.6 Å. Scale bars: 10 μm in B–C, 5 μm in E–F, 500 nm in G.







Schematic diagrams of a Chlamydomonas cell and its phototactic behavior.
(Top) The eyespot is located near the cell equator and contains the carotenoid granule layers (red) and photoreceptor proteins, channelrhodopsins (ChR1 and ChR2; blue). The carotenoid layers reflect a light beam (orange arrows) and amplify the light signal from the outside of the cell on ChR (the “front side”) and block the light from the inside of the cell (the “rear side”). The flagellum closest to the eyespot is called the cis-flagellum, whereas the other one is called the trans-flagellum. Modified from refs. 24 and 41. (Bottom) As the cell swims with self-rotation, the eyespot apparatus scans the incident light around the cell’s swimming path. After photoreception by the channelrhodopsins, the cell changes the beating balance of the two flagella and exhibits either positive or negative phototaxis (swimming toward or away from the light source, respectively). 15



(1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; 
(2) the primary function of Type B sensory rhodopsin (channel-rhodopsin-2) is biochemical activation of secondary Ca2+-channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; 
(3) Type A sensory rhodopsin (channelrhodopsin-1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low-efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells. 16 Striking features of photosensory reception in flagellates are: (1) its extremely high, nearly single-quantum, sensitivity, and (2) very large dynamic intensity range of motility responses. The presented data show that this combination is achieved by dividing the ~6 orders of magnitude light intensity range in which the responses can be elicited between two transduction pathways of dual-function sensory rhodopsins.




 


1. http://www.ijdb.ehu.es/web/descarga/paper/041900wg.
2. http://www.cell.com/cell/pdf/S0092-8674(15)01565-2.pdf
3. http://www.cell.com/cell/pdf/S0092-8674(11)01502-9.pdf
4. https://wikivisually.com/wiki/Eyespot_apparatus
4. Evolution of Visual and Non-visual Pigments, page 110
5. http://rstb.royalsocietypublishing.org/content/364/1531/2819
6. https://www.researchgate.net/publication/23689154_The_green_algal_eyespot_apparatus_A_primordial_visual_system_and_more
7. https://sci-hub.bz/http://www.annualreviews.org/doi/full/10.1146/annurev.arplant.59.032607.092847
8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1533972/
9. https://answersingenesis.org/biology/microbiology/volvox-single-celled-synchronized-swimmers/
10. https://www.nature.com/scitable/topicpage/volvox-chlamydomonas-and-the-evolution-of-multicellularity-14433403
11. https://www.biologie.hu-berlin.de/de/gruppenseiten/expbp/research/channelrhodopsins-light-activated-ion-channels
12. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868408/
13, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483371/
14. https://elifesciences.org/articles/04889
15. https://sci-hub.bz/http://science.sciencemag.org/content/357/6356/eaan5544
16. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622041/

Carotenoid biosynthesis: 
Carotenoids in Algae: Distributions, Biosyntheses and Functions
Mevalonate pathway

Among the proteins are five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot.  About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody.  68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca2+-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii. Furthermore: calcium-sensing and binding proteins, channels, membrane-associated/structural proteins such as proteins with PAP-fibrillin domains, proteins involved in retinal, carotenoid, and chlorophyll biosynthesis as well as in lipid metabolism, and thylakoid membrane-associated proteins. Of special interest is also the presence of a SOUL heme-binding protein (SOUL/HBP) in the eyespot. The first member of this group of HBPs was found to be specifically located in the retina and pineal gland in chicken. Kinases and phosphatases were found in the eyespot proteome, indicating that light signaling cascades(s) may involve regulation by reversible protein phosphorylation. The two detected protein phosphatases (PPs) belong to the PP2C family of Ser/Thr PPs. The five identified kinases in the eyespot proteome include a cyclic nucleotide-dependent kinase II, two unusual protein kinases with AarF domains, the blue light photoreceptor phototropin with its Ser/Thr kinase domain, and casein kinase1 (CK1).


Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation 1




1. https://www.frontiersin.org/articles/10.3389/fpls.2015.01085/full

Origin of eyespots - supposedly one of the simplest eyes

The Evolution of the Eye, Demystified Otangelo Grasso February 24, 2020, 4:08 AM
https://evolutionnews.org/2020/02/the-evolution-of-the-eye-demystified/?fbclid=IwAR3fgWoc02Xy0jgggCKa2TLLofsQkuSh3vUWf2xSNk73jU1iMJGzwiTJk2Y

1. Rhodopsin, the first player of vision, is a light-sensitive receptor protein involved in visual phototransduction. Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. If both were not just right from the beginning, rhodopsin would not be functional, and vision, not possible. Retinal is finely tuned and right-sized to fit the binding pocket. It is a molecule obtained by a complex multistep biosynthesis pathway.
2. Both, opsin, and retinal, are highly constrained and thus well conserved across organisms because mutations within these sites are deleterious.
3. An article in Nature magazine, in 2016, confessed: Darwin’s original puzzle over ocular evolution seems still to be with us but now at a molecular level.
4. The inference is that Rhodopsin is irreducibly complex, and its origin, therefore, best explained by design!

https://reasonandscience.catsboard.com/t2638-volvox-eyespots-and-interdependence#5768

JM’s first nonsense, doing the work in the melting snow. Precisely when work can’t be done. Would you work in freezing cold water wetting your clothes the entire time you work and deadly rocks regularly falling.


Rhodopsins  are expressed in a specialized compartment - the so-called eyespot- where light initiates a fast inward-directed photocurrent. The electrical signal is amplified by the activation of voltage-gated secondary channels and is transmitted to the two flagella which in turn adjust their beating plane, frequency and pattern. Hence, the complex interplay of photoreceptors and flagella movement enables the algae to perform positive or negative phototaxis according to the quality of the ambient light. 1

Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. embed in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein. 3

Nilsson's famous paper on eye evolution starts with an eyespot, and in a nice row of pictures shows how eyespots could have evolved to complex camera eyes : 



Representative stages of a model sequence of eye evolution. 
In the initial stage (1) the structure is a flat patch of light-sensitive cells sandwiched between a transparent protective layer and a layer of dark pigment  1

In the last sentence of the paper, which has been used since it was published in 1994 as a reference to back up the claim of the evolution of  eyes, Nilsson writes :

" the eye was never a real threat to Darwin's theory of evolution."

In the article  Light and the evolution of vision in Nature magazine, the author writes: 
Chlamydomonas is green algae in the plant kingdom. Phototaxis is essential for it; moving towards light upon which they depend for energy and nutrition, yet also undergoing negative phototaxis to protect themselves against too intense a source of illumination. The eyespot is not the photoreceptor itself but rather a mass of carotenoid pigment shading the photoreceptor from light from one direction. This demonstrates the essential components of any visual system; any photosensitive organism needs a photoreceptor that detects the light. But that alone would not allow the organism to determine the direction of the light source. A pigment spot reduces the illumination from one direction, or changes the wavelength of the incident light falling on the photoreceptor, thus allowing the organism to move in the direction of the light or away for it. So third, a mechanism to promote movement is essential. To detect the light is one thing but to move towards or away from it requires a motor system; the flagellae in Chlamydomona. But also a mechanism is required by which detection of light can be translated into a change in flagellar movement, generally an ion flux of one kind or another.

This demonstrates the essential components of this visual system:

1. any photosensitive organism needs a photoreceptor that detects the light. 
2 A pigment spot reduces the illumination from one direction, or changes the wavelength of the incident light falling on the photoreceptor, thus allowing the organism to move in the direction of the light or away for it.
3  a mechanism to promote movement is essential.  it requires a motor system; the flagellae
4- But also a mechanism is required by which detection of light can be translated into a change in flagellar movement, generally an ion flux of one kind or another. Trans-membrane calcium flux initiates a cascade of electrical responses causing depolarization of the cell and ultimately controls the flagellar beating pattern. 




This is an interdependent system composed of 4 essential components, photoreceptors, a pigment spot, the flagellae, and ion flux, of which, if one is missing, the organism cannot move by phototaxis. Natural selection would not select any intermediate evolutionary step, since the system, with any of the four elements missing, would confer no function, and no advantage of survival.  

From the kinetics and the rhodopsin action spectrum of these photocurrents we conclude that they are part of the rhodopsin-regulated signal transduction chain controlling the cellular behaviour in light. Both photocurrents are Ca²⁺-dependent and are suppressed by the Ca²⁺-channel inhibitors verapamil and pimozide, suggesting that the photoreceptor current and probably the flagellar current are both carried by Ca²⁺. 5




Question: Why did the authors begin their narrative with a " flat patch of light-sensitive cells "? rather than  with an explanation of how such a "patch" could have evolved? and what it would be good for, unless being there for a specific function, like vision, detection of light/shading, circadian rhythm etc., which always requires other parts that constitute a system of at least two parts?  

The claim is that a unicellular, "simple" organism, like Volvox green algae, may have been equipped with a photoreceptor organelle, and the eyes may have evolved from such an ancestral state. The story of proponents of the evolution of the eye goes as follows: The simple light-sensitive spot on the skin of some ancestral creature gave it some tiny survival advantage, perhaps allowing it to evade a predator. 1

There is IMHO nothing " simple ": eyespots like in Chlamydomonas reinhardtii algae, have 742 different proteins 8 ; they have an elaborate structure, and a  high ultrastructural complexity 18  .     
Zoologist Dan-Erik Nilsson avoided asking the relevant question: What good is an eyespot for by itself? 

From the "simplest", most rudimentary eye forms, like eyespots,  to complex vertebrate eyes, like our camera eyes, rhodopsins are the first players in a complex chain of biochemical events. In unicellular organisms, like Chlamydomonas, eyespots shade dark from the light and interconnected with the flagellum, they either distance from clarity, or move closer to sunlight, depending on their needs. This is an interdependent system, where one has no function unless linked to the other.  

Rhodopsin is the central player in vision. There is no vision without it. Unless rhodopsin transforms light into a signal, and that signal is used by a signal transduction pathway to promote phototaxis, neither rhodopsins nor eyespots would bear function by their own. A flagellum cannot rotate to move the cell in the right direction unless it gets the right instructions. 

The design of the eyespot apparatus in conjunction with the helical movement of the cell produces a highly directional optical device allowing effective tracking of the light direction. In Chlamydomonas reinhardtii, the eyespot apparatus is usually composed of two layers of highly ordered carotenoid-rich lipid globuli that are situated at the periphery of the chloroplast. The globuli layers are subtended by thylakoid membranes. Additionally, the outermost globule layer is attached to specialized areas of the chloroplast envelope membranes and the adjacent plasma membrane.

The carotenoid layers reflect a light beam and amplify the light signal from the outside of the cell on rhodopsin (the “front side”) and block the light from the inside of the cell (the “rear side”).  These carotenoid granules are crucial for phototaxis. Crucial = indispensable - irreducible. As the cell swims with self-rotation, the eyespot apparatus scans the incident light around the cell’s swimming path. After photoreception by the rhodopsins, the cell changes the beating balance of the two flagella and exhibits either positive or negative phototaxis (swimming toward or away from the light source, respectively).

The phototactic pathway primarily consists of four steps: 

(i) photoreception by Channel rhodopsins
(ii) excitation of the cellular membrane; 
(iii) increase in intraflagellar [Ca2+]; and 
(iv) a change in the beating balance between the two flagella, i.e., the cis-flagellum (the one closest to the eyespot) and the trans-flagellum (the one farthest from the eyespot)


Important cellular processes result from the concerted action of multiple proteins organized in complex networks. Studies in evolution have revealed how individual proteins can acquire new functions due to changes in their binding specificity or catalytic potential. However, these characteristics alone often cannot explain the evolution of complex cellular functions, because network output does not solely depend on the function of an individual protein, but rather on the integrated function of multiple components with intricate regulatory relationships. 2

This is what we observe even in unicellular organisms like Chlamydomonas where motility depends on a multitude of different proteins interconnected and highly regulated to adapt to the different environmental conditions.

Rhodopsin Structure and Activation
Channelrhodopsins, the first players of vision,  are expressed in a specialized compartment - the so-called eyespot- where light initiates a fast inward-directed photocurrent. The electrical signal is amplified by the activation of voltage-gated secondary channels and is transmitted to the two flagella which in turn adjust their beating plane, frequency and pattern. Hence, the complex interplay of photoreceptors and flagella movement enables the algae to perform positive or negative phototaxis according to the quality of the ambient light. 1

Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. embed in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein. 3  

Channelrhodopsin has only function conjoined with retinal. 

Evolution of Rhodopsins
Opsins are a group of proteins that underlie the molecular basis of various light sensing systems including phototaxis, circadian (daily) rhythms, eye sight, and a type of photosynthesis. Opsins are  retinal proteins because they bind to a light-activated, non-protein chromophore called retinal. All opsin proteins are embedded in cell membranes, crossing the membrane seven times. 6

Functional residues, such as those within the catalytic sites of enzymes, are highly constrained and thus well conserved across organisms because mutations within these sites are normally deleterious. 3

That raises the question how these G Proteins emerged in the first place since they are highly specific and prone to mutations. 

An often cited source of evolutionary novelty is the recruiting and co-option of extant building blocks, and incorporate them into new systems, by natural selection of new functions. Rhodopsin would have to undergo evolution by recruiting All-trans-retinal chromophores, which it would have to find ready fully formed and functional, and finely tuned and right-sized to fit the binding pocket,  a molecule obtained by a complex multistep biosynthesis pathway starting with carotenoid chromophores from fruits, flowers, trees or vegetables. 4  It would require elaborated import mechanisms and the information how to insert it in the binding pocket, and attached at the right place, and the insertion of a protonated retinal Schiff base  ( The term Schiff base is normally applied to these compounds when they are being used as ligands ) 

The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. 14

The binding of the chromophore to the opsin is essential to trigger the conformational change and must be precise and functional from the beginning. Following is required :  

1. a Schiff base linkage
2. a Lys296 residue where chromophore retinal covalently binds
3. the side chain of the residue
4. an essential amino acid residue called "counter ion". The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. 17
5. There is a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of  rhodopsin

Unless all of these specific points were not just right from the beginning, rhodopsin would not be functional. Each of these processes demands already coordinated and finetuned interplay and precise orchestration between opsin and retinal. 

Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105

Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if subsystems or parts are put together in the right order, they also need to interface correctly.


The question is: How did opsins and their configuration of seven precisely folded alpha helix transmembranes emerge? 

Rao et. al. have proposed that "...the packing of seven helices together may represent a uniquely stable arrangement that has been achieved through a process of convergent evolution." 10

Here we go. We " have proposed ".... convergent evolution. But but.... where is the evidence ??

In the paper: The Origins of Novel Protein Interactions during Animal Opsin Evolution, the authors make the remarkable admittance: 

Genetic changes are known to modify phenotype during evolution by altering the interactions between a protein and its ecological or biochemical environment, by modulating existing protein-protein interaction. However, the specific genetic changes that give rise to the evolutionary origins of novel protein-protein interactions HAVE RARELY BEEN DOCUMENTED IN DETAIL.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001054

Origin of correct protein folding, a major problem in evolutionary biology

The precision upon which opsins must fold into their seven transmembrane configuration is staggering: 

Biophysicists at JILA have measured protein folding in more detail than ever before, revealing behavior that is surprisingly more complex than previously known. . . .2 the JILA team identified 14 intermediate states—seven times as many as previously observed—in just one part of bacteriorhodopsin, a protein in microbes that converts light to chemical energy and is widely studied in research. “The increased complexity was stunning,” said project leader Tom Perkins, a National Institute of Standards and Technology (NIST)  “Better instruments revealed all sorts of hidden dynamics that were obscured over the last 17 years when using conventional technology.” “If you miss most of the intermediate states, then you don’t really understand the system,” he said. Knowledge of protein folding is important because proteins must assume the correct 3-D structure to function properly. Misfolding may inactivate a protein or make it toxic. Several neurodegenerative and other diseases are attributed to incorrect folding of certain proteins. 
https://www.nist.gov/news-events/news/2017/03/jila-team-discovers-many-new-twists-protein-folding

An article in Nature magazine confirms :
even as far back as the prokaryotes the complex seven transmembrane domain arrangement of opsin molecules seems to prevail without simpler photoreceptors existing concurrently. Darwin’s original puzzle over ocular evolution seems still to be with us but now at a molecular level. 4
https://www.nature.com/articles/eye2015220



Retinal chromophores: 
channelrhodopsin-1 and channelrhodopsin-2 (ChR-1 and ChR-2), are directly light-gated cation channels that contain a planar all-trans, 6-S-trans retinal chromophore, which undergoes 13-trans to cis isomerization upon illumination.

Retinal is a unique molecule with a chemical design that allows optimal interaction with the opsin apoprotein in its binding pocket, and this is essential for the formation of the light-activated conformation of the receptor. 2  When a photon strikes this retinal chromophore and the light energy is absorbed by retinal, this light energy is used to cause one of the alkenes in retinal to undergo a  configuration change. This configuration change causes a change in the conformation (the three-dimensional shape) of the opsin protein , which triggers the complex transduction cascade.  



All structural details in the retinal chromophore are functionally important 

A paper reports an intriguing evolutionary conservation of the key components involved in chromophore production and recycling. The synthesis of retinal precedes a complex pathway of several enzymatic steps starting from carotenoids molecules. There would have been no evolutionary advantage to evolve such a pathway and its proteins, unless there was the know how to make the molecule with the correct structure, in order to work fine and fit correctly in the opsin pocket to form a functional Rhodopsin protein. 


Carotenoids biosynthesis in Chlamydomonas reinhardtii





Schematic diagram of the carotenoid biosynthetic pathway in plants and microalgae. 

Phytoene synthase (PSY) catalyses the first step in the carotenoid specific pathway, which leads the carbon flux towards carotenes and xantophylls production. 
IPP isopentenyl pyrophosphate, 
DMAPP dimethylallyl pyrophosphate, 
GGPP geranylgeranyl pyrophosphate, 
GGPPS geranylgeranyl pyrophosphate synthase, 
PDS phytoene desaturase, 
Z-ISO 15-cis-ζ-carotene isomerase, 
ZDS ζ-carotene desaturase, CRTISO carotene isomerase, 
LCYb lycopene β-cyclase, 
LCYe lycopene ε-cyclase, 
P450b-CHY cytochrome P450 β-hydroxylase, 
P450e-CHY cytochrome P450 ε-hydroxylase, 
CHYb carotene β-hydroxylase, 
BKT β-carotene oxygenase, 
ZEP zeaxanthin epoxidase, 
violaxanthin de-epoxidase

Retinal, the chromophore that is covalently linked to the rhodopsin-type photoreceptors of the eyespot apparatus likely results from symmetric cleavage of β-carotene by a β -carotene-15,15  -oxygenase (BCO). candidate genes related to the animal enzyme have been identified in Chlamydomonas

We have by this only scratched the surface. We would have to explain the precise interplay and complex mechanism between the eyespot and the flagella, describe the flagellum in all its complexity. But what has been demonstrated so far is, that eyespots are FAR FROM simple, and depend on many interdependent parts, which, if not fully interconnected and regulated, would not permit Volvox to swim either towards the light (positive phototaxis) or away from the light.  

We can safely say: Vision and its origin is best explained by intelligent design

The eyespot plays an accessory role in photobehavioral responses and eyeless mutant would be able to perceive and respond to light. Recent study using the reactive oxygen species (ROS), leads to the identification of novel eyeless mutant of Chlamydomonas exhibiting strong phototaxis responses. These reports support the earlier made hypothesis, that the photoperception in Chlamydomonas is not confined to eyespot. One possibility is that the bacterial rhodopsins localized in the flagella of Chlamydomonas might be responsible for a non-directional phototransduction of this organism. The eyespot-guided phototaxis is very important for the zooplankton larvae of marine invertebrates and is proposed to mediate larval swimming towards the light. Recently, it has been proposed that eyespot localized opsin of the Platynereis are the photoreceptors for controlling phototaxis of this organism. It would be interesting to elucidate the role of intraflagellar transport (IFT) machinery in the trafficking of opsin in the eyespot of the Platynereis, which would shed light on evolutionary link of the of the IFT mediated trafficking of the rhodopsin(s) in nature. The involvement of IFT in the eyespot localization of rhodopsin would further support its role in intracellular trafficking of proteins similar to the case of immune synapse assembly in higher animals.

1. http://faculty.jsd.claremont.edu/dmcfarlane/bio145mcfarlane/PDFs/Nilsson%20and%20Pelger_eye%20evolution%20model%20ProcRoyalSoc_1994.pdf.
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976203/
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177186/
4. http://www.nature.com/eye/journal/v30/n2/full/eye2015220a.html
5. https://sci-hub.tw/https://www.nature.com/nature/journal/v351/n6326/abs/351489a0.html

The trafficking of bacterial type rhodopsins into the Chlamydomonas eyespot and flagella is IFT mediated 1

Here we present the first report for the existence of an IFT-interactome mediated trafficking of the bacterial type rhodopsins into eyespot and flagella of the Chlamydomonas. We show that there is a light-dependent, dynamic localization of rhodopsins between flagella and eyespot of Chlamydomonas. light-regulated localization of rhodopsin is not restricted to animals thereby suggesting that rhodopsin trafficking is an IFT dependent ancient process. The placement of rhodopsins at the proper site in the cell is crucial for optimal photoperception and the subsequent photoresponses. Lower motile organisms like the unicellular algae, Chlamydomonas reinhardtii, possess bacterial type rhodopsins in the eyespot for photosensing. The non-motile organisms have spectrally tuned rhodopsin to match the wavelength of light abundant in their niche.

IFT is a highly orchestrated and dedicated means of protein transport in the cilia/flagella. The assembly, maintenance and functioning of these sensory organelles require IFT. In IFT, These motor proteins in association with IFT particles, carry some of the ciliary cargoes but some ciliary cargoes are known to be carried independent of these motor proteins. Defects in the IFT and ciliogenesis are linked with many developmental disorders and diseases collectively referred to as ciliopathies. The ciliopathies related to rhodopsin trafficking lead to defects like impaired vision, irreversible blindness, Retinitis Pigmentosa, Leber Congenital Amarouses.

Chlamydomonas possesses seven different bacterial type rhodopsins called chlamyopsins. Chlamyopsin 3 and 4 (Cop3 and Cop4) are involved in the photo-behavioral (phototaxis and photophobic) responses and because of their light-activated ion channel activities, these have been renamed as channelrhodopsin 1 (ChR1)and channelrhodopsin 2 (ChR2), respectively. Channelrhodopsins mediate photoreceptor current in the eyespot and also trigger the flagellar photocurrent that in turn brings about the change in calcium flux across the membrane. Trans-membrane calcium flux initiates a cascade of electrical responses causing depolarization of the cell and ultimately controls the flagellar beating pattern. Another photoreceptor protein (phototropin) has been recently observed to influence eyespot development, ChR1 regulation and phototactic behavior. Studies related to the cellular localization of ChR1 showed that channelrhodopsins are localized in the eyespot of Chlamydomonas with the help of cytoskeletal components. However, how Chlamydomonas rhodopsins are trafficked inside the cell and how this transport is regulated are largely unknown.

This report provides the first evidence for the involvement of intraflagellar transport (IFT) in the ferrying of bacterial type rhodopsin proteins. IFT molecular motors and IFT particles were found to be involved in the trafficking of Chlamyopsin8/Cop8 (novel rhodopsin identified in this study) and ChR1 into the flagella, in a light dependent manner. Use of different conditional IFT mutants enabled us to monitor the fate of Cop8 and ChR1 in IFT depleted yet flagellated Chlamydomonas cells. The interaction studies provided the evidences of the interaction between Chlamydomonas rhodopsins and the components of IFT machinery along with the proteins involved in the IFT-cargo complex formation. Our data leads to a model in which IFT machinery participates in the rhodopsin transport in unicellular eukaryotic green algae Chlamydomonas reinhardtii. It suggests that IFT mediated trafficking of rhodopsin is not only restricted to vertebrates but also occurs in lower eukaryotes.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5046144/

Origin of eyespots - supposedly one of the simplest eyes

https://reasonandscience.catsboard.com/t2638-volvox-eyespots-and-interdependence#5768

Nilsson's famous paper on eye evolution published in 1994 serves still today as reference science article in regards to how the eye evolved and is often cited by proponents of evolution. It starts with an eyespot, and in a nice row of pictures shows how eyespots could have evolved to complex camera eyes.  In the initial stage, the structure is a flat patch of light-sensitive cells sandwiched between a transparent protective layer and a layer of dark pigment  

In the last sentence of the paper, Nilsson writes : " the eye was never a real threat to Darwin's theory of evolution."

In the article  Light and the evolution of vision in Nature magazine, the author writes: 
Chlamydomonas is green algae in the plant kingdom. Phototaxis is essential for it; moving towards light upon which they depend for energy and nutrition, yet also undergoing negative phototaxis to protect themselves against too intense a source of illumination. The eyespot is not the photoreceptor itself but rather a mass of carotenoid pigment shading the photoreceptor from light from one direction. This demonstrates the essential components of any visual system; any photosensitive organism needs a photoreceptor that detects the light. But that alone would not allow the organism to determine the direction of the light source. A pigmented spot reduces the illumination from one direction or changes the wavelength of the incident light falling on the photoreceptor, thus allowing the organism to move in the direction of the light or away from it. So third, a mechanism to promote movement is essential. To detect the light is one thing but to move towards or away from it requires a motor system; the flagella in Chlamydomonas. But also a mechanism is required by which detection of light can be translated into a change in flagellar movement, generally an ion flux of one kind or another.

This demonstrates the essential components of this visual system:

1. any photosensitive organism needs a photoreceptor that detects the light. 
2 A pigmented spot reduces the illumination from one direction or changes the wavelength of the incident light falling on the photoreceptor, thus allowing the organism to move in the direction of the light or away for it.
3  a mechanism to promote movement is essential.  it requires a motor system; the flagellae
4- But also a mechanism is required by which detection of light can be translated into a change in flagellar movement, generally an ion flux of one kind or another. Trans-membrane calcium flux initiates a cascade of electrical responses causing depolarization of the cell and ultimately controls the flagellar beating pattern. 

This is an interdependent system composed of 4 essential components, photoreceptors, a pigment spot, the flagella, and ion flux, of which, if one is missing, the organism cannot move by phototaxis. Natural selection would not select any intermediate evolutionary step, since the system, with any of the four elements missing, would confer no function, and no advantage of survival.  


Question: Why did the authors begin their narrative with a " flat patch of light-sensitive cells "? rather than with an explanation of how such a "patch" could have evolved? and what it would be good for, unless being there for a specific function, like vision, detection of light/shading, circadian rhythm etc., which always requires other parts that constitute a system of at least two parts?  

The claim is that a unicellular, "simple" organism, like Volvox green algae, may have been equipped with a photoreceptor organelle, and the eyes may have evolved from such an ancestral state. The story of proponents of the evolution of the eye goes as follows: The simple light-sensitive spot on the skin of some ancestral creature gave it some tiny survival advantage, perhaps allowing it to evade a predator. 1

There is nothing " simple ": eyespots like in Chlamydomonas reinhardtii algae, have 742 different proteins 8 ; they have an elaborate structure, and a  high ultrastructural complexity 18  .     
Zoologist Dan-Erik Nilsson avoided asking the relevant question: What good is an eyespot for by itself? 

From the "simplest", most rudimentary eye forms, like eyespots,  to complex vertebrate eyes, like our camera eyes, rhodopsins are the first players in a complex chain of biochemical events. In unicellular organisms, like Chlamydomonas, eyespots shade dark from the light and interconnected with the flagellum, they either distance from clarity, or move closer to sunlight, depending on their needs. This is an interdependent system, where one has no function unless linked to the other.  

Rhodopsin is the central player in vision. There is no vision without it. Unless rhodopsin transforms light into a signal, and that signal is used by a signal transduction pathway to promote phototaxis, neither rhodopsins nor eyespots would bear function by their own. A flagellum cannot rotate to move the cell in the right direction unless it gets the right instructions. 





The design of the eyespot apparatus in conjunction with the helical movement of the cell produces a highly directional optical device allowing effective tracking of the light direction. In Chlamydomonas reinhardtii, the eyespot apparatus is usually composed of two layers of highly ordered carotenoid-rich lipid globuli that are situated at the periphery of the chloroplast. The globuli layers are subtended by thylakoid membranes. Additionally, the outermost globule layer is attached to specialized areas of the chloroplast envelope membranes and the adjacent plasma membrane.

The carotenoid layers reflect a light beam and amplify the light signal from the outside of the cell on rhodopsin (the “front side”) and block the light from the inside of the cell (the “rear side”).  These carotenoid granules are crucial for phototaxis. Crucial = indispensable - irreducible. As the cell swims with self-rotation, the eyespot apparatus scans the incident light around the cell’s swimming path. After photoreception by the rhodopsins, the cell changes the beating balance of the two flagella and exhibits either positive or negative phototaxis (swimming toward or away from the light source, respectively).

The phototactic pathway primarily consists of four steps: 

(i) photoreception by Channel rhodopsins
(ii) excitation of the cellular membrane; 
(iii) increase in intraflagellar [Ca2+]; and 
(iv) a change in the beating balance between the two flagella, i.e., the cis-flagellum (the one closest to the eyespot) and the trans-flagellum (the one farthest from the eyespot)


Important cellular processes result from the concerted action of multiple proteins organized in complex networks. Studies in evolution have revealed how individual proteins can acquire new functions due to changes in their binding specificity or catalytic potential. However, these characteristics alone often cannot explain the evolution of complex cellular functions, because network output does not solely depend on the function of an individual protein, but rather on the integrated function of multiple components with intricate regulatory relationships. 2

This is what we observe even in unicellular organisms like Chlamydomonas where motility depends on a multitude of different proteins interconnected and highly regulated to adapt to the different environmental conditions.

Rhodopsin Structure and Activation
Channelrhodopsins, the first players of vision,  are expressed in a specialized compartment - the so-called eyespot- where light initiates a fast inward-directed photocurrent. The electrical signal is amplified by the activation of voltage-gated secondary channels and is transmitted to the two flagella which in turn adjust their beating plane, frequency and pattern. Hence, the complex interplay of photoreceptors and flagella movement enables the algae to perform positive or negative phototaxis according to the quality of the ambient light. 1

Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. embed in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein. 3  

Channelrhodopsin has only function conjoined with retinal. 

Evolution of Rhodopsins
Opsins are a group of proteins that underlie the molecular basis of various light sensing systems including phototaxis, circadian (daily) rhythms, eye sight, and a type of photosynthesis. Opsins are  retinal proteins because they bind to a light-activated, non-protein chromophore called retinal. All opsin proteins are embedded in cell membranes, crossing the membrane seven times. 6

Functional residues, such as those within the catalytic sites of enzymes, are highly constrained and thus well conserved across organisms because mutations within these sites are normally deleterious. 3

That raises the question how these G Proteins emerged in the first place since they are highly specific and prone to mutations. 

An often cited source of evolutionary novelty is the recruiting and co-option of extant building blocks, and incorporate them into new systems, by natural selection of new functions. Rhodopsin would have to undergo evolution by recruiting All-trans-retinal chromophores, which it would have to find ready fully formed and functional, and finely tuned and right-sized to fit the binding pocket,  a molecule obtained by a complex multistep biosynthesis pathway starting with carotenoid chromophores from fruits, flowers, trees or vegetables. 4  It would require elaborated import mechanisms and the information how to insert it in the binding pocket, and attached at the right place, and the insertion of a protonated retinal Schiff base  ( The term Schiff base is normally applied to these compounds when they are being used as ligands ) 

The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. 14

The binding of the chromophore to the opsin is essential to trigger the conformational change and must be precise and functional from the beginning. Following is required :  

1. a Schiff base linkage
2. a Lys296 residue where chromophore retinal covalently binds
3. the side chain of the residue
4. an essential amino acid residue called "counter ion". The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. 17
5. There is a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of  rhodopsin

Unless all of these specific points were not just right from the beginning, rhodopsin would not be functional. Each of these processes demands already coordinated and finetuned interplay and precise orchestration between opsin and retinal. 

Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105

Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if subsystems or parts are put together in the right order, they also need to interface correctly.


The question is: How did opsins and their configuration of seven precisely folded alpha helix transmembranes emerge? 

Rao et. al. have proposed that "...the packing of seven helices together may represent a uniquely stable arrangement that has been achieved through a process of convergent evolution." 10

Here we go. We " have proposed ".... convergent evolution. But but.... where is the evidence ??

In the paper: The Origins of Novel Protein Interactions during Animal Opsin Evolution, the authors make the remarkable admittance: 

Genetic changes are known to modify phenotype during evolution by altering the interactions between a protein and its ecological or biochemical environment, by modulating existing protein-protein interaction. However, the specific genetic changes that give rise to the evolutionary origins of novel protein-protein interactions HAVE RARELY BEEN DOCUMENTED IN DETAIL.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001054



Origin of correct protein folding, a major problem in evolutionary biology

The precision upon which opsins must fold into their seven transmembrane configuration is staggering: 

Biophysicists at JILA have measured protein folding in more detail than ever before, revealing behavior that is surprisingly more complex than previously known. . . .2 the JILA team identified 14 intermediate states—seven times as many as previously observed—in just one part of bacteriorhodopsin, a protein in microbes that converts light to chemical energy and is widely studied in research. “The increased complexity was stunning,” said project leader Tom Perkins, a National Institute of Standards and Technology (NIST)  “Better instruments revealed all sorts of hidden dynamics that were obscured over the last 17 years when using conventional technology.” “If you miss most of the intermediate states, then you don’t really understand the system,” he said. Knowledge of protein folding is important because proteins must assume the correct 3-D structure to function properly. Misfolding may inactivate a protein or make it toxic. Several neurodegenerative and other diseases are attributed to incorrect folding of certain proteins. 
https://www.nist.gov/news-events/news/2017/03/jila-team-discovers-many-new-twists-protein-folding

An article in Nature magazine confirms :
even as far back as the prokaryotes the complex seven transmembrane domain arrangement of opsin molecules seems to prevail without simpler photoreceptors existing concurrently. Darwin’s original puzzle over ocular evolution seems still to be with us but now at a molecular level. 4
https://www.nature.com/articles/eye2015220

Retinal chromophores: 
channelrhodopsin-1 and channelrhodopsin-2 (ChR-1 and ChR-2), are directly light-gated cation channels that contain a planar all-trans, 6-S-trans retinal chromophore, which undergoes 13-trans to cis isomerization upon illumination.

Retinal is a unique molecule with a chemical design that allows optimal interaction with the opsin apoprotein in its binding pocket, and this is essential for the formation of the light-activated conformation of the receptor. 2  When a photon strikes this retinal chromophore and the light energy is absorbed by retinal, this light energy is used to cause one of the alkenes in retinal to undergo a  configuration change. This configuration change causes a change in the conformation (the three-dimensional shape) of the opsin protein , which triggers the complex transduction cascade.  All structural details in the retinal chromophore are functionally important.  

A paper reports an intriguing evolutionary conservation of the key components involved in chromophore production and recycling. The synthesis of retinal precedes a complex pathway of several enzymatic steps starting from carotenoids molecules. There would have been no evolutionary advantage to evolve such a pathway and its proteins, unless there was the know how to make the molecule with the correct structure, in order to work fine and fit correctly in the opsin pocket to form a functional Rhodopsin protein. 

Retinal, the chromophore that is covalently linked to the rhodopsin-type photoreceptors of the eyespot apparatus likely results from symmetric cleavage of β-carotene by a β -carotene-15,15  -oxygenase (BCO). candidate genes related to the animal enzyme have been identified in Chlamydomonas

We have by this only scratched the surface. We would have to explain the precise interplay and complex mechanism between the eyespot and the flagella, describe the flagellum in all its complexity. But what has been demonstrated so far is, that eyespots are FAR FROM simple, and depend on many interdependent parts, which, if not fully interconnected and regulated, would not permit Volvox to swim either towards the light (positive phototaxis) or away from the light.  

We can safely say: Vision and its origin is best explained by intelligent design

The eyespot plays an accessory role in photobehavioral responses and eyeless mutant would be able to perceive and respond to light. Recent study using the reactive oxygen species (ROS), leads to the identification of novel eyeless mutant of Chlamydomonas exhibiting strong phototaxis responses. These reports support the earlier made hypothesis, that the photoperception in Chlamydomonas is not confined to eyespot. One possibility is that the bacterial rhodopsins localized in the flagella of Chlamydomonas might be responsible for a non-directional phototransduction of this organism. The eyespot-guided phototaxis is very important for the zooplankton larvae of marine invertebrates and is proposed to mediate larval swimming towards the light. Recently, it has been proposed that eyespot localized opsin of the Platynereis are the photoreceptors for controlling phototaxis of this organism. It would be interesting to elucidate the role of intraflagellar transport (IFT) machinery in the trafficking of opsin in the eyespot of the Platynereis, which would shed light on evolutionary link of the of the IFT mediated trafficking of the rhodopsin(s) in nature. The involvement of IFT in the eyespot localization of rhodopsin would further support its role in intracellular trafficking of proteins similar to the case of immune synapse assembly in higher animals.

Opsin by opsin, and then came the retinal, and last not least, the rhodopsin. Molecule by molecule, slowly out of the dim and out of the gloom somehow, someway, one day, mutation by mutation, trial and error here and there, alleles by frequency and change, without direction, but slowly and through lots and lots of time, natural selection selecting here, and a bit there,  the eyespot apparatus made its debut on the scene of daylight. Amazing feat. One, two, three, but hey, eyes, yet, couldn't see, what happened ??!! Our newly born photoreceptors were terribly alone and without use. ahhh.... I see. There was no connection. But, hey, that is no problem, after all, time and a bit evolution make all great and purposeful things happen. Chlamydomonas, our not yet seeing green algae was also terribly bored by not exploring the great big pond and sea, and not beating, swimming, so, the flagella made its debut and came up on the scene.  It wiggled and began its job of amazing propulsion, but Chlamy was swimming like silly and drunken, without direction. Something had to happen... Brilliant idea !! Why not form a connection. And then there will be direction. And so it was. The photoreceptor and the flagella got connected, a new amazing invention debuted on the scene. Calcium signalling from the eye to the flagella was invented by evolution and bingo!! Our irreducibly complex combo of eye, calcium signalling communication, and beating of the flagella in an orderly fashion, and obeying the right direction of Chlamy: phototaxis was born. Now, it could swim, by daylight go down, by night go up on the waterline. What an amazing feat. Aren't nature and natural selection great partners & friends ?!! What a wonderful world.....

Origin of eyespots - supposedly one of the simplest eyes

Nilsson's famous paper on eye evolution starts with an eyespot, and in a nice row of pictures shows how eyespots could have evolved to complex camera eyes : 



The claim is that an unicellular, "simple" organism, like Volvox, may have been equipped with a photoreceptor organelle, and the eyes may have evolved from such an ancestral state. The story of proponents of the evolution of the eye goes as follows: The simple light-sensitive spot on the skin of some ancestral creature gave it some tiny survival advantage, perhaps allowing it to evade a predator. 1

The earliest structure remotely related to an eye is the eyespot, a light sensing structure in the green alga Chlamydomonas. 21

The two-cell eyespot of this marine invertebrate is one of the simplest animal eyes and consists of a photoreceptor cell and a pigmented cell that shades the photoreceptor, defining its view angle. 19

The simplest animal eyes are eyespots composed of two cells only: a photoreceptor and a shading pigment cell. They resemble Darwin’s ‘proto-eyes’, considered to be the first eyes to appear in animal evolution  20 We propose that the underlying direct coupling of light sensing and ciliary locomotor control was a principal feature of the proto-eye and an important landmark in the evolution of animal eyes. 

There is IMHO nothing " simple ": eyespots like in Chlamydomonas reinhardtii algae, have 202 different proteins 8; they have an elaborate structure, and a  high ultrastructural complexity 18  .     

Zoologist Dan-Erik Nilsson missed however to ask the relevant question: What good is an eyespot by itself? 

From the "simplest", most rudimentary eye forms, like eyespots,  to complex vertebrate eyes, like our camera eyes, rhodopsins are the first players in a complex chain of biochemical events. In unicellular organisms, like Chlamydomonas, eyespots shade dark from the light and interconnected with the flagellum, they either distance from clarity, or move closer to sunlight, depending on their needs. This is an interdependent system, where one has no function unless linked to the other.  So there is another example of INTERDEPENDENCE.

Rhodopsin is the central player in vision. We can safely say, there is no vision without it. Unless rhodopsin transforms light into an electrical signal, and that signal is used by a signal transduction pathway to promote phototaxis, neither rhodopsins nor eyespots would bear function by their own.  That already constitutes an INTERDEPENDENT system.  A flagellum cannot rotate to move the cell in the right direction unless it gets the instruction in which direction to move, which comes from the eyespot and signal transduction. 





Functional categorization and characterization of identified phosphoproteins from the eyespot fraction 18

Photoreceptors
ChR-1 precursors
Retinal-binding protein
Retina-related proteins
SOUL/HBP
MORN repeat protein 1
Kinases
Predicted unusual protein kinase (ISS)
Ser/Thr protein kinase stt7
Calcium-binding protein
Similar to calmodulin 2
Protein with PAP-fibrillin domain
NCBI BLASTp, PAP-fibrillin domain
Carotenoid and fatty acid metabolism-related proteins
1-Deoxy-d-xylulose-5-P synthase
Sterol-C-methyltransferase
Omega-3 fatty acid desaturase
Thylakoid and chloroplast membrane-related proteins
Hypothetical protein [Chlamydomonas sp. HS-5], PSII 10-kD polypeptide PsbR
Tic62 protein
PSI reaction center subunit VI, chloroplast precursor (PSI-H); (LHCI, 11-kD protein; P28 protein)
Chloroplastic outer envelope membrane protein (OEP75)
LhcbM8, LhcbM4, LhcbM6; LHCII protein precursors
Cytochrome b6-f complex subunit petO, phosphoprotein precursor
Kelch repeat: Kelch (ISS)
S-adenosyl-Met synthetase
Cobalamin-dependent Met synthase (ISS)
Os10g0455900
Heat shock protein 70B
Contaminants
Putative RNA helicase (ISS)
External rotenone-insensitive NADPH dehydrogenase (ISS)
mRNA- processing factor 3 domain
Conserved and novel proteins of yet unknown function
6 different proteins of unknown function

The design of the eyespot apparatus in conjunction with the helical movement of the cell produces a highly directional optical device allowing effective tracking of the light direction. In Chlamydomonas reinhardtii, the eyespot apparatus is usually composed of two layers of highly ordered carotenoid-rich lipid globuli that are situated at the periphery of the chloroplast. The globuli layers are subtended by thylakoid membranes. Additionally, the outermost globule layer is attached to specialized areas of the chloroplast envelope membranes and the adjacent plasma membrane.

The eyespot contains carotenoid granule layers beside photoreceptor proteins. 



The carotenoid layers reflect a light beam and amplify the light signal from the outside of the cell on rhodopsin (the “front side”) and block the light from the inside of the cell (the “rear side”).  These carotenoid granules are crucial for phototaxis. Crucial = indispensable - irreducible. As the cell swims with self-rotation, the eyespot apparatus scans the incident light around the cell’s swimming path. After photoreception by the rhodopsins, the cell changes the beating balance of the two flagella and exhibits either positive or negative phototaxis (swimming toward or away from the light source, respectively).

The phototactic pathway primarily consists of four steps: 

(i) photoreception by Channel rhodopsins
(ii) excitation of the cellular membrane; 
(iii) increase in intraflagellar [Ca2+]; and 
(iv) a change in the beating balance between the two flagella, i.e., the cis-flagellum (the one closest to the eyespot) and the trans-flagellum (the one farthest from the eyespot)

Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions 6
Important cellular processes result from the concerted action of multiple proteins organized in complex networks. Studies in evolution have revealed how individual proteins can acquire new functions due to changes in their binding specificity or catalytic potential. However, these characteristics alone often cannot explain the evolution of complex cellular functions, because network output does not solely depend on the function of an individual protein, but rather on the integrated function of multiple components with intricate regulatory relationships. 

This is what we observe even in unicellular organisms like Chlamydomonas where motility depends on a multitude of different proteins interconnected and highly regulated to adapt to the different environmental conditions.

Rhodopsin Structure and Activation
Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. Rhodopsins are light-sensitive  G protein–coupled receptors (GPCRs) that embed in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein. 3  Channelrhodopsin has only function conjoined with retinal. 



Origin of correct protein folding, a major problem in evolutionary biology
An often cited source of evolutionary novelty is the recruiting and co-option of extant building blocks, and incorporate them into new systems, by evolution of new functions. Rhodopsin would have to undergo evolution by recruiting All-trans-retinal Chromophores, which it would have to find ready, and finely tuned and right-sized to fit the binding pocket,  a molecule obtained by a fully developed multistep biosynthesis pathway starting with carotenoid chromophores from fruits, flowers, trees or vegetables. 4  It would require complex import mechanisms and the information how to insert it in the binding pocket, and attached at the right place, forming a protonated retinal Schiff base  ( The term Schiff base is normally applied to these compounds when they are being used as ligands ) 



The Schiff base linkage between 13-cis retinal and the epsilon-amino group of lys216. During the photocycle, this base gets protonated and deprononated as part of the proton-pumping process.

The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. 14



Eukaryotic G Protein Signaling Evolved to Require G Protein-Coupled Receptors for Activation  8
Functional residues, such as those within the catalytic sites of enzymes, are highly constrained and thus well conserved across organisms because mutations within these sites are normally deleterious.

That raised the question how these G Proteins emerged in the first place since they are highly specific and prone to mutations.  

We extend this concept of constraint from the level of the primary sequence to the functional traits of signaling proteins, and we propose that an intrinsic functional property of a signaling molecule, which is often not evident in 3D
structures, is also evolutionally constrained by the binding regulatory partners;

This means, there is a configuration, that is just right, between interacting apoproteins and cofactors, and constrained like lock and key. Not any  will do, but the form must be precise and fit correctly, to confer function. 

Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if subsystems or parts are put together in the right order, they also need to interface correctly. 9
They continue: Once a protein-protein interaction is fixed, the molecular trait (for example, a binding surface) becomes constrained.

They have jumped the relevant part: How did the trait arise in the first place ??!!

Here, we showed that the origins of the GEF-dependent G protein correlated with the expansion in the number of GPCR-encoding genes

So, the authors of this paper do explain basically nothing. They just claim that the number of genes expanded, but how, remains unanswered, and is hidden by all the " sciency" jargon...  

We have already seen that rhodopsin is a bundle of seven transmembrane helices. This structural feature can also be found in other integral membrane receptor proteins, such as those of the adrenergic, cholinergic, serotonin, somatostatin, and other receptor families. Rao et. al. have proposed that "...the packing of seven helices together may represent a uniquely stable arrangement that has been achieved through a process of convergent evolution." 10

Here we go again. " have proposed ".... convergent evolution. But but.... where is the evidence ??

The specific genetic changes that give rise to the evolutionary origins of novel protein-protein interactions have rarely been documented in detail 11
Opsins comprise two protein families, called type I and type II opsins, with detailed functional similarities, but several lines of evidence indicate that the two opsin classes evolved separately, illustrating an amazing case of convergent evolution. 13

Type I and type II rhodopsins share several structural features including a G protein-coupled receptor fold and a highly conserved active-site Lys residue in the seventh transmembrane segment of the protein. However, the two families lack significant sequence similarity that would indicate common ancestry. Consequently, the rhodopsin fold and conserved Lys are widely thought to have arisen from functional constraints during convergent evolution. 14

The evolutionary relation between two large groups of sensory membrane proteins, namely the G-protein coupled receptors (GPCRs) and microbial rhodopsins (MRs) has been puzzling biologists for almost four decades. 
Opsins comprise two protein families, called type I and type II opsins, with detailed functional similarities, but several lines of evidence indicate that the two opsin classes evolved separately, illustrating an amazing case of convergent evolution.

Type I and type II rhodopsins share several structural features including a G protein-coupled receptor fold and a highly conserved active-site Lys residue in the seventh transmembrane segment of the protein. However, the two families lack significant sequence similarity that would indicate common ancestry. 15 Consequently, the rhodopsin fold and conserved Lys are widely thought to have arisen from functional constraints during convergent evolution.

Not only is the explanation of convergence made up just so. But, I would say, this is rather an amazing case of DESIGN !! If getting seven functional transmembranes with the extremely fine-tuned amino acid sequence to get the right fold and form a correctly sized, finely tuned binding pocket for a retinal chromophore that fits in just right and interacts just right, is already an amazing feat, but imagine TWICE by different routes and amino acid sequences !! And getting the active-site Lys residue in the seventh transmembrane segment of the protein TWICE is hardly well explained by " functional constraints ", if rhodopsin would become only functional once the binding site is extant and retinal as well and able to bind to it. 

Perhaps the Lys has been retained in TM7 because neither of these intermediates is capable of forming a viable pigment.

So opsins had a pre-established goal, and worked by trial and error until the set goal would have been achieved to form a viable pigment? That teleology at its best... 

What was said about Bacteriorhodopsin, can certainly be said as well about Channelrhodopsins, which share the same architecture, namely:

Biophysicists at JILA have measured protein folding in more detail than ever before, revealing behavior that is surprisingly more complex than previously known. . . .2 the JILA team identified 14 intermediate states—seven times as many as previously observed—in just one part of bacteriorhodopsin, a protein in microbes that converts light to chemical energy and is widely studied in research. “The increased complexity was stunning,” said project leader Tom Perkins, a National Institute of Standards and Technology (NIST)  “Better instruments revealed all sorts of hidden dynamics that were obscured over the last 17 years when using conventional technology.” “If you miss most of the intermediate states, then you don’t really understand the system,” he said. Knowledge of protein folding is important because proteins must assume the correct 3-D structure to function properly. Misfolding may inactivate a protein or make it toxic. Several neurodegenerative and other diseases are attributed to incorrect folding of certain proteins.

In metazoans, these trends are strongest in tissues composed of neurons, whose structure and lifetime confer extreme sensitivity to protein misfolding.

The binding of the chromophore to the opsin is essential to trigger the conformational change. That means, there had to be

1. a Schiff base linkage
2. a Lys296 residue where chromophore retinal covalently binds
3. the side chain of the residue
4. an essential amino acid residue called "counter ion"
5. There is a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of  rhodopsin

Retinal chromophores: 
channelrhodopsin-1 and channelrhodopsin-2 (ChR-1 and ChR-2), are directly light-gated cation channels that contain a planar all-trans, 6-S-trans retinal chromophore, which undergoes 13-trans to cis isomerization upon illumination.



All structural details in the retinal chromophore are functionally important 

1. https://www.pbs.org/wgbh/evolution/library/01/1/l_011_01.html
2. https://uncommondescent.com/intelligent-design/discovery-of-7-times-higher-complexity-of-protein-folding/
3. https://en.wikipedia.org/wiki/Rhodopsin
4. https://www.ch.ic.ac.uk/vchemlib/mim/bristol/retinal/retinal_text.htm
5. https://www.openoptogenetics.org/index.php?title=Channelrhodopsins
6. https://www.nature.com/articles/ncomms12344
7. https://biologydirect.biomedcentral.com/articles/10.1186/s13062-015-0091-4
8. https://sci-hub.bz/http://stke.sciencemag.org/content/6/276/ra37
9. ( Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105 (Rowman & Littlefield, 2004). HT: ENV.)
10. http://www.cryst.bbk.ac.uk/PPS95/us/iddo/badstruc.htm
11. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001054
12. Encyclopedia of the eye, 4th edition, page 1469
13. http://evolutionarynovelty.blogspot.com.br/2008/12/opsins-amazing-evolutionary-convergence.html
14. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746867/
15. http://www.pnas.org/content/110/33/13351.full
16. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245826/
17. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245826/table/tbl1/[/b][/b]
18. https://www.frontiersin.org/articles/10.3389/fpls.2015.01085/full
19. http://stke.sciencemag.org/content/1/47/ec402
20. https://sci-hub.bz/http://www.nature.com/nature/journal/v456/n7220/abs/nature07590.html
21. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546623/

Rhodopsins falsify the claim of evolution of the eye

Channelrhodopsins are the first and most essential and important players of vision. They are expressed in a specialized compartment - the so-called eyespot- where light initiates a fast inward-directed photocurrent. The electrical signal is amplified by the activation of voltage-gated secondary channels and is transmitted to the two flagella which in turn adjust their beating plane, frequency and pattern. Hence, the complex interplay of photoreceptors and flagella movement enables the algae to perform positive or negative phototaxis according to the quality of the ambient light.
Rhodopsin consists of two components, a protein molecule called opsin and a covalently-bound cofactor called retinal. embed in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein.  

Channelrhodopsin has only function by the interplay conjoined with retinal, and as such, they are interdependent. 

Evolution of Rhodopsins
Opsins are a group of proteins that underlie the molecular basis of various light sensing systems including phototaxis, circadian (daily) rhythms, eyesight, and a type of photosynthesis. Opsins are retinal proteins because they bind to a light-activated, non-protein chromophore called retinal. All opsin proteins are embedded in cell membranes, crossing the membrane seven times.  Functional residues, such as those within the catalytic sites of enzymes, are highly constrained and thus well conserved across organisms because mutations within these sites are normally deleterious. 

That raises the question of how these precursors of opsins,  G Proteins, emerged in the first place since they are highly specific and prone to mutations. 

An often cited source of evolutionary novelty is the recruiting and co-option of extant building blocks, and incorporate them into new systems, by natural selection of new functions. Rhodopsin would have to undergo evolution by recruiting All-trans-retinal chromophores, which it would have to find ready fully formed and functional, and finely tuned and right-sized to fit the binding pocket. Retinal is  a molecule obtained by a complex multistep biosynthesis pathway starting with carotenoid chromophores from fruits, flowers, trees or vegetables.  It would require elaborated import mechanisms and the information how to be inserted it in the binding pocket, and attached at the right place, and the insertion of a protonated retinal Schiff base ( The term Schiff base is normally applied to these compounds when they are being used as ligands ) 

The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. 

The binding of the chromophore to the opsin is essential to trigger the conformational change which in sequence triggers the signal transduction pathway, which transmits a signal to the brain, ion channels for flagella beating etc. and must be precise and functional from the beginning. Following is required :  

1. a Schiff base linkage
2. a Lys296 residue where chromophore retinal covalently binds
3. the side chain of the residue
4. an essential amino acid residue called "counter ion". The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. 17
5. There is a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of  rhodopsin

Unless all of these specific points were not just right from the beginning, rhodopsin would not be functional. Each of these processes demands already coordinated and finetuned interplay and precise orchestration between opsin and retinal. 

We read in the book: Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105
Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if subsystems or parts are put together in the right order, they also need to interface correctly.

The first question is: How did opsins and their configuration of seven precisely folded alpha helix transmembranes emerge, and later, the precise binding sites for retinal, our second essential player in sight? 

Rao et. al. have proposed that "...the packing of seven helices together may represent a uniquely stable arrangement that has been achieved through a process of convergent evolution." 

Here we go. We " have proposed ".... convergent evolution. But but.... where is the evidence ??

In the paper: The Origins of Novel Protein Interactions during Animal Opsin Evolution, the authors make the remarkable admittance: 

Genetic changes are known to modify phenotype during evolution by altering the interactions between a protein and its ecological or biochemical environment, by modulating existing protein-protein interaction. However, the specific genetic changes that give rise to the evolutionary origins of novel protein-protein interactions HAVE RARELY BEEN DOCUMENTED IN DETAIL.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001054

The origin of correct protein folding is a major problem in evolutionary biology. The precision upon which opsins must fold into their seven transmembrane configurations is staggering: 

Biophysicists at JILA have measured protein folding in more detail than ever before, revealing behavior that is surprisingly more complex than previously known. . . .2 the JILA team identified 14 intermediate states—seven times as many as previously observed—in just one part of bacteriorhodopsin, a protein in microbes that converts light to chemical energy and is widely studied in research. “The increased complexity was stunning,” said project leader Tom Perkins, a National Institute of Standards and Technology (NIST) “Better instruments revealed all sorts of hidden dynamics that were obscured over the last 17 years when using conventional technology.” “If you miss most of the intermediate states, then you don’t really understand the system,” he said. Knowledge of protein folding is important because proteins must assume the correct 3-D structure to function properly. Misfolding may inactivate a protein or make it toxic. Several neurodegenerative and other diseases are attributed to incorrect folding of certain proteins. 
https://www.nist.gov/news-events/news/2017/03/jila-team-discovers-many-new-twists-protein-folding

An article in Nature magazine confirms :
Even as far back as the prokaryotes the complex seven transmembrane domain arrangement of opsin molecules seems to prevail without simpler photoreceptors existing concurrently. Darwin’s original puzzle over ocular evolution seems still to be with us but now at a molecular level. 
https://www.nature.com/articles/eye2015220

This is a KEY-confession which basically puts to an end all claims that the evolution of the eye is a solved problem, and that the evidence has been found. 
 
Retinal 
Channelrhodopsins are directly light-gated channels that contain retinal, which undergoes 13-trans to cis isomerization upon illumination. Is a unique molecule with a chemical design that allows optimal interaction with the opsin protein in its binding pocket, and this is essential for the formation of the light-activated conformation of the receptor. When a photon strikes this retinal chromophore and the light energy is absorbed by retinal, this light energy is used to cause one of the alkenes in retinal to undergo a  configuration change. This configuration change causes a change in the conformation (the three-dimensional shape) of the opsin protein , which triggers the complex transduction cascade.  All structural details in the retinal chromophore are functionally important. 

A paper reports an intriguing evolutionary conservation of the key components involved in chromophore production and recycling. The synthesis of retinal precedes a complex pathway of several enzymatic steps starting from carotenoids molecules. There would have been no evolutionary advantage to evolve such a pathway and its proteins, unless there was the know how to make the molecule with the correct structure, in order to work fine and fit correctly in the opsin pocket to form a functional Rhodopsin protein. 

Metazoan opsin evolution reveals a simple route to animal vision
https://www.pnas.org/content/109/46/18868?fbclid=IwAR1LSVGslcIj2HTrY7a-jDzCfv1BnsQbKwXhCkwI6CkhXXmDWB8Kz84-SLM

Conclusions
The 11-cis-retinal chromophore is covalently attached to the protein by means of a protonated Schiff base to the ε-amino group of Lys296 in the seventh helix. The GPCR fold and active-site Lys are absolutely conserved among all visual pigments of higher eukaryotes.

Scarcity of signal for the deepest event in the history of the opsin family implies that some level of uncertainty in opsin evolution still remains, and might be unavoidable., and
the K296-based retinal binding domain ( RBD ) most likely evolved, probably through a process of neofunctionalization. ".

Reply: Following paper :  

Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins
https://www.pnas.org/content/110/33/13351

states:  
In the present study, we attempted to move the Schiff base Lys from position 296 in TM7 to position 117 in TM3, but the mutant is incapable of forming a pigment with added 11-cis-retinal
The K296G and K296A mutant rhodopsins, in which the active-site Lys has been removed, cannot covalently bind 11-cis-retinal.
Relocating the Lys would likely require an intermediate either with no active-site Lys or with Lys residues at both locations. Perhaps the Lys has been retained in TM7 because neither of these intermediates is capable of forming a viable pigment.

Bovine rhodopsin exists physiologically as ahomodimer consisting of 349 amino acids each and lies near the middle of the spectrum of polypeptide chain lengths among the GPCR family.

That means, natural selection would have had to find the Lys 296 position amongst an average of 349 amino acids to bind retinal in order to bear function. But it would have also require to bind functional retinal, which is highly specific. As following paper states:

A methyl group at C7 of 11-cis-retinal allows chromophore formation but affects rhodopsin activation
https://www.sciencedirect.com/science/article/pii/S0042698906003580

Retinal is a unique molecule with a chemical design that allows optimal interaction with the opsin apoprotein in its binding pocket, and this is essential for the formation of the light-activated conformation of the receptor. When a photon strikes this retinal chromophore and the light energy is absorbed by retinal, this light energy is used to cause one of the alkenes in retinal to undergo a  configuration change. This configuration change causes a change in the conformation (the three-dimensional shape) of the opsin protein , which triggers the complex transduction cascade.

Having both just right, that is, the right binding position, and retinal in its functional form, is already a huge challenge. But on top of that, retinal has to be recycled

Intelligent Design And The Origin Of The Visual Cycle
https://uncommondescent.com/intelligent-design/intelligent-design-and-the-origin-of-the-visual-cycle/

Continuous vision depends on recycling of the photoproduct all-trans-retinal back to visual chromophore 11-cis-retinal. This process is enabled by the visual (retinoid) cycle, a series of biochemical reactions in photoreceptor, adjacent RPE and Müller cells.

Evolution and the origin of the visual retinoid cycle in vertebrates
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781855/

The origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery.

Claim:
And the paper that you think shows the lysine residue must be located at K296 actually does not show that.

Reply:
The paper shows, that the residue cannot be placed at any position, and that Lys residues are necessary in order for retinal to bind to the protein. To find, select and bind to one of the 11 Lys residues amongst ~350 amino acids by unguided random happenstance is already a considerable feat and constraint.  

the paper states:
" We show here that the Lys can be moved to three other locations in the protein while maintaining the ability to form a pigment with 11-cis-retinal and activate the G protein transducin in a light-dependent manner.

In figure 1 of the paper, it can be observed, that Lys was inserted in adjacent positions at surrounding helices near the 296 location.

That indicates, if retial would have been connected to any Lys residue further away from the binding pocket, there would be no function.  

They move on:
" These results demonstrate that an absolutely conserved, common structural feature—the Schiff base Lys in helix seven—is not required for rhodopsin’s photosensitive function, contradicting a key prediction of convergent evolution resulting from functional constraint."

If evolutionary predictions were met, any of the few mentioned functional positions could have been selected, which, IMHO, is not the case. So to explain that, another ad hoc assertion is provided:

" These results contradict the convergent hypothesis and support the homology of type I and type II rhodopsins by divergent evolution from a common ancestral protein."

Divergent evolution is the process whereby groups from the same common ancestor evolve and accumulate differences, resulting in the formation of new species. Convergent evolution means that the same function was reached by independent evolutionary routes, which theoretically would be the case here.

The paper claims that convergent evolution did not result from functional constraint. So what was it?

If selecting a functional position, aka Lys296, or any of the few possible positions that bear function, would have been already a considerable feat, what to say about two homologous but amino-acid sequentially divergent proteins bearing the same function, that supposedly evolved in a divergent but also convergent manner from a common ancestor, selecting exactly the same Lys296 position ??!!

Claming that " a process of neofunctionalization did it " is an ad-hoc assertion that actually does not explain anything. Why would/should evolutionary pressures cause that neofunctionalization ? Bear in mind that:

- retinal is only functional if just right in its form
- retinal must be recycled , and if the full irreducible multiprotein process is not fully set up, no deal. Retinal is not recycled, no vision is the result.
- retinal is a Vitamin A, and its biosynthesis is an extremely complex multistep process requiring several proteins, and that has also to be explained. Forsight of the end function seems to be necessary in order to produce a molecule which fits perfectly in the opsin pocket, know how in which binding positions it bears function, and how to recycle it.
- rhodopsin by itself would have no function, unless it is embedded in a process like locomotion, where visual information is required for the bacteria to move closer, or more far away from sunlight.  

All this seems to be only eloquently explained by intentional design through the action of an extremely intelligent designer with foresight.