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Origin of phototransduction, the visual cycle, photoreceptors and retina

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Origin of phototransduction, the visual cycle, photoreceptors and retina

http://reasonandscience.heavenforum.org/t1638-origin-of-phototransduction-the-visual-cycle-photoreceptors-and-retina

Links to the main topics below:

Evolutionary proposals of phototransduction
The phototransduction pathway - how it works
Rhodopsin , opsins , and retinol
Type 1 Opsins
The Visual Cycle
Retinal
Cone and Rod photoreceptor cells

Study suggests humans can detect even the smallest units of light
'Any man-made detector would need to be cooled and isolated from noise to behave the same way.” 4

The first true understanding of how the vertebrate eye works came in the early seventeenth century, with mathematician Johannes Kepler’s demonstration that vision occurs as an image projected on to the surface of the retina 5

Study suggests humans can detect even the smallest units of light – July 21, 2016
Excerpt: Research,, has shown that humans can detect the presence of a single photon, the smallest measurable unit of light. Previous studies had established that human subjects acclimated to the dark were capable only of reporting flashes of five to seven photons.,,,
it is remarkable: a photon, the smallest physical entity with quantum properties of which light consists, is interacting with a biological system consisting of billions of cells, all in a warm and wet environment,” says Vaziri. “The response that the photon generates survives all the way to the level of our awareness despite the ubiquitous background noise. Any man-made detector would need to be cooled and isolated from noise to behave the same way.”,,,
The gathered data from more than 30,000 trials demonstrated that humans can indeed detect a single photon incident on their eye with a probability significantly above chance.
“What we want to know next is how does a biological system achieve such sensitivity? How does it achieve this in the presence of noise?

The eye can potentially be exposed to an enormous range of light intensities, ranging from a few photons per second under extremely dim lighting conditions to light levels that can be more than 10-billion-fold higher (i.e., a factor of 10^10 or 10000000000 log units). Furthermore, there may be rapid temporal fluctuations in the light level due to eye movements. In addition, there can be marked changes in the chromatic properties of the visual environment, such as when viewing objects in incandescent room illumination versus outdoors at noon on a sunny day. Remarkably, the visual system is able to cope with the large range of illumination conditions through complex neural mechanisms that are collectively termed adaptation.

Each human eye has about one hundred twenty million rods arranged throughout the retina. The rods contain a photopigment called rhodopsin which is very sensitive to all the wavelengths of the visible light spectrum. In contrast, there are only about six million cones that are mostly concentrated in the macula, primarily in the cone-only fovea. Each cone contains one of three different photosensitive pigments, called photopsins, which tend to react stronger to either the red, green, or blue wavelengths of light. Both rhodopsin and the photopsins are dependent on Vitamin A. 3

When photons of light strike the retina they interact with the photoreceptor cells and cause an electrical change and the release of a neurotransmitter.

The Comb Jelly Opsins and the Origins of Animal Phototransduction 2

Early opsin evolution has yet to be fully understood, in large part because of the high level of divergence observed among opsins belonging to different subfamilies. 2 Opsins are key to understanding the origins and evolution of light sensitivity, eyes, and vision. Many hypotheses of opsin evolution have been proposed, but consensus has remained elusive.

In particular, two recent studies analyzed complementary data sets, reaching very dissimilar conclusions with conflicting implications for opsin origins, and our understanding of early animal evolution

The first study by Feuda et al. (2012) found sequences from Placozoa (that they called “placopsins”) to be the sister of all known animal opsins, and consistent with other studies, they found melatonin receptors to be the closest outgroup to opsins + placopsins. Placopsins remain functionally uncharacterized, and because they lack the retinal-binding lysine, they might not function in light reception.

The scenario proposed by Feuda et al. (2012) to explain their results suggests that visual opsins evolved after Placozoa separated from Cnidaria and Bilateria but before the latter separated from each other. Feuda et al. (2012) did not have data for Ctenophora (i.e., the comb jellies). However, given previous phylogenomic results suggesting that Ctenophora, Cnidaria, and Bilateria are more closely related with each other than they are with the sponges and the Placozoa, they concluded that their results were compatible with a traditional view of animal evolution (an hypothesis we refer to as “Neuralia”). Differently from Nielsen (2012), Neuralia is here to be interpreted as simply stating that Bilateria, Cnidaria, and Ctenophora shared a common ancestor to the exclusion of the Placozoa and the sponges, irrespective of whether, within Neuralia, Cnidaria and Ctenophora form monophyletic Coelenterata or a paraphyletic group where Ctenophora is closer to Bilatera than it is to Cnidaria.

Animal vision evolved 700 million years ago 1

Every ray of light that you perceive was caught by an opsin first. Without opsin there would be no blue, no red, no green. The entire visible spectrum would be.. just another spectrum. 1  Opsin is a member of large family of detector proteins, called the 'G-protein coupled receptors' (GPCRs). Like a needle and thread, all GPCRs wind themselves through the outer membrane of the cell seven times. Halfway between cell and outside world, these tiny sensors are perfectly positioned to monitor the surroundings of the cell. Most GPCRs detect the presence of certain molecules. When a certain hormone or neurotransmitter docks their outward facing side they become activated and release signalling molecules on the inside of the cell. But opsin is different. It doesn't bind molecules physically. Instead, it senses the presence of a more delicate and ephemeral particle: the photon itself, the particles (and waves) that light is made of.  

Opsins trap photons with a small molecule in the heart of their architecture, called retinal. In its resting state retinal has a bent and twisted tail. But as soon as light strikes retinal, its tail unbends. This molecular stretching exercise forces the opsin to change shape as well. The opsin is now activated and eventually will cause a nearby nerve to fire, which will relay its message to the brain: light!.

First of all, Feuda confirmed the existence of three distinct opsin types within bilateria (bilaterians are animals with left-right symmetry). These three opsin types are called R-opsins, C-opsins and RGR-opsins. For a long time biologists thought C-opsins were exclusively found in animals with a spine (the vertebrates) and that R-opsins were limited to protostomes, a diverse group of animals that includes mollusks and arthropods. (The third type of opsin, the RGR-opsin, is a bit odd compared to the other opsins. Instead of detecting light, they play a role in regenerating 'spent' retinal molecules.)

The division was so stark and neat that vertebrates and protostomes must each have evolved their own light detecting opsins from an ancestral template. Or so scientists thought. The tidy story unraveled once opsins started to pop up in unsuspected places. The brain of the ragworm Platynereis dunerlii, a protostome, was found to contain C-opsins. R-opsins were identified in nerve cells in the human retina. These discoveries forced opsin biologists back to the evolutionary drawing board. In their new scenario, the common ancestor of vertebrates and protostomes, the ur-bilaterian, already had three types of opsin. The two lineages later recruited C-opsins or R-opsins for their visual systems, respectively.

Sure enough, the placozoan genome harbours two opsins. But here's the catch: these opsins cannot detect light. Remember retinal, the molecule that changes shape when it is struck by light? The placozoan opsins cannot bind retinal, because they lack the amino acid to which retinal binds (amino acids are the building blocks of proteins). Without 'lysine-296', it is unlikely that the placozoan opsins can detect light. But if not light sensors, what then? "Surely placozoans use these opsins. How? I cannot tell.

It's remarkable, how evidence is interpreted however someone can imagine to fit the evolutionary framework !! No evidence on the contrary refutes evolution. No matter what !!

Yet Feuda's results bear one mind-boggling implication: the c-opsins in your cones and rods are more closely related to the corresponding opsins in the eye-spots of a jellyfish, than either of them is to the r-opsin in your retinal nerves.  To see how deep, Feuda's team leapt to another branch of the family tree, and scoured the genomes of two sponges, Oscarella and Amphimedon, for opsin sequences. No dice. Apparently, opsins only evolved after sponges had diverged from other animals, but before the split between Bilateria and Cnidaria. Fortunately for Feuda, there exists one animal lineage in this sweet spot between sponges on one side and cnidarians/bilaterians on the other. Meet the placozoans. Small, simple and flat, placozoans resemble shapeshifting pancakes more than anything else. They drift along the sea floor, searching for detritus to scavenge.

Pondering this figure, it hit me that our opsins really had two origins. One is the birth of opsin itself, the other is the mutation that turned opsin into a light sensing protein. The opsin lineage itself arose between 755 and 711 million years ago, from the duplication of a single GPCR. The last common ancestor of Bilateria and Cnidaria lived between 711 and 700 million years ago. This leaves a short window of time (evolutionary speaking) in which opsin acquired the light sensing mutation and split into the three opsin families we still carry today.

How did natural mechanisms be able to produce a retinol molecule which Photon absorption isomerizes the retinal from the bent 11-cis form to the all-trans conformation, and do so with blazing speed--it only takes a few hundred femtoseconds (10-13 sec).? If the isomerization would not take place, no conformational change, no signal transduction, and vision would not have appeared on earth in biological organisms.....  the point is, retinol has no function unless bound on the seventh alpha helice on a lysine amino acid of opsin. If not doing so, it will not transmit and signal of conformational change to opsin, and its isomerization is useless. So - no opsin, no use of retinol...... and vice versa. No retinol, no use for opsin. both depend on the other in order for signal transduction to begin - and there is a further chain of events, which depend on essential enzymes, proteins, and membrane channels to produce a signal, which will be transmitted in a complex way to where it will be processed.

Absorption of a photon by a rhodopsin or cone-opsin pigment induces isomerization of its retinal chromophore, activating the signal transduction pathway , which will transmit a visual information to the brain. Once the retinal chromophore is isomerized to all-trans conformation, it's bleached and  its light sensitivity requires to be  restored by chemical reisomerization  via a multistep enzyme pathway, called the visual cycle. This occurs partially in  cells of the retinal pigment epithelium (RPE).   

Further readings:

The Evolution Of The Visual System In Primates
http://redwood.berkeley.edu/bruno/animal-eyes/Kaas_revised_2013.pdf

http://darwins-god.blogspot.com.br/search?q=eye
Charles Darwin considered the eye to be an “organ of extreme perfection.

Eye evolution and its functional basis
Shedding new light on opsin evolution
The opsins
Evolution of Phototransduction, Vertebrate Photoreceptors and Retina
THE EVOLUTION OF OPSINS AND COLOR VISION
Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup
Metazoan opsin evolution reveals a simple route to animal vision
Chemistry and Biology of Vision
Fine tuning of Light, to Atmosphere, Water, Photosynthesis, and Human Vision

1. https://blogs.scientificamerican.com/thoughtomics/animal-vision-evolved-700-million-years-ago/
2. https://academic.oup.com/gbe/article/6/8/1964/568182/The-Comb-Jelly-Opsins-and-the-Origins-of-Animal
3. https://evolutionnews.org/2016/08/the_mystery_of/
4. http://phys.org/news/2016-07-humans-smallest.html
5. https://sci-hub.bz/http://www.nature.com/nature/journal/v523/n7559/full/nature14630.html



Evolutionary proposals  of opsins, and phototransduction 

The phylogenetic relationship between the basic opsin-classes appears difficult to resolve 3

We can presume that rhodopsin(s) had a very early origin, going back at least to the common ancestor of Archaebacteria and Eukaryotes, since these proteins are present in both these superkingdoms. 4   It is unlikely that this protein evolved more than once in nature. Many of these macromolecules are so specific and unique for their ability to carry on a particular function, as rhodopsin does in the photoreceptive process, to be present in animal and plant kingdoms every time this specific function is demanded. 

It is well in agreement with the calculate estimate of microspectrophotometric data 1.5.10^7, or 15.000.000 molecules of rhodopsin per cell 6 and with the fact that in photoreceptor structures the number of molecules is about 10^7-10^8. The data we collected from our experiments allowed us to state the rhodopsin nature of Euglena photoreceptor. Our results can be considered to be in agreement with the evidence presented in 1991 by Hegemann et al. , by Derguini et al. and by Kreimer et al. that all-trans-retinal is present in the photoreceptive organelles of other microalgae, namely Chlamydomonas and Spermatozopsis.

The actual reported numbers are ≈10^5 rhodopsins per membrane disc or ≈10^8 per photoreceptor (BNID 108323), which is on the order of the total number of proteins expected for such cell volume as discussed in the vignette on “How many proteins are in a cell?”. This tight packing is what enables the eye to be able to function so well at extremely low light levels. 5



Given the outer segment ≈25,000 nm length, this means that there are roughly 1000 such discs in each of the ≈108 rod cells in the vertebrate retina (with about 108 rhodopsin molecules per rod cell as discussed in the vignette on “How many rhodopsin molecules are in a rod cell?”). These 1000 effective layers increase the cross section available for intercepting photons making our eyes such “organs of extreme perfection”. 


The origin of photoreceptor cells indicated in Fig. below 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

One of the first steps in eye evolution must have been the appearance of a light-dependent chemical reaction coupled to a signaling system. 19

This alone would already consist in a unbridgeable hurdle for unguided mechanisms like natural selection. It's irrelevant, how simple such a system would be. It would already consist of a system essentially interlinked in two functional parts, where one depends on the other. Signal systems do depend, without exception, on the setup of the signal code or language,  and correct understanding and common agreement of the sender-receiver, a system that can only be set up by conscient intelligent beings. 

Light-dependent reactions are always very specific, and from an engineering standpoint, very demanding, complex, precise,, and depend on the correct arrangement of protein-protein interactions, enzymatic reactions which need to be finely tuned, and implemented with specificity.
 

Evolutionary Origins of Phototransduction 18

At some time before the divergence of jellyfish and humans, but likely after the split common ancestor of sponges and humans, the first light sensitive animal opsin protein originated. This protein did not originate from nothing, nor was it newly breathed into an ancient animal genome by a designer. Instead, opsins arose by mutation of an existing receptor to render it light sensitive

Really ?!!

In the case the opsins in question, scientists have determined the entire genome sequence of the sponge Amphimedon queenslandica, the choanoflagellate Monosiga and numerous fungi, making the presence of opsin in those organisms very unlikely. Some proteins of near-animals are in fact rather similar to opsins, but in every case the non-animal receptors lack characteristics that specifically define opsins. Therefore, although opsins might have been present at the origin of animals and lost in sponges, their absence instead strongly suggests that they originated within animals, before the split common ancestor of humans, insects, and cnidarians, all of which possess opsins.

Opsins form a subfamily within a larger family of proteins called G-protein coupled receptors (GPCRs), also sometimes called serpentine proteins because they snake back and forth across cell membranes. Since serpentine proteins are present in all animals and their close relatives - including sponges, Monsiga, and fungi - this broad class of proteins long predates animals. In yeast (a fungus), these receptors are sensitive to pheromones and they even direct a signal through proteins homologous to non-opsin phototransduction proteins. As such, a signaling pathway exists outside animals, which is very similar to phototransduction, except the receptor protein detects pheromones, not light.

Darwin’s question can be refined farther, to “how did a serpentine protein gain the ability to respond to light?” And since opsin’s light sensitivity is mediated by its ability to bind a light reactive chemical, called a chromophore, the question can be even further refined to ask how a GPCR must be modified to bind a chromophore. In the case of opsin, we know that a particular amino acid – a Lysine in the 7th membrane-spanning region – binds to the light reactive chemical. Presumably then, a mutation changing an amino acid in the 7th trans-membrane region of a light-insensitive GPCR was involved in the acquisition of light sensitivity in animals. This fateful mutation coupled with numerous other mutations, are responsible for the origins of eyes and vision.

Well, what had to be adquired, is a chromophore. But not just any kind of chromophore. But one that could be recyled after bleaching through a complex visual cycle. 

1

General scheme of eye evolution.
The first step in eye evolution is the evolution of a light receptor molecule which in all metazoa is rhodopsin. In the most ancestral metazoa, the sponges, a single Pax gene, but no opsin gene has been found. In the cubozoan jellyfish Tripedalia, a unicellular photoreceptor has been described in the larva. The adult jellyfish has complex lens eyes which form under the control of PaxB, whereas the eyes of a hydrozoan jellyfish (Cladonema) are controlled by PaxA. We propose that from the unicellular photoreceptor cell the prototypic eye postulated by Darwin originated by a first step of cellular differentiation into photoreceptor cell and pigment cell controlled by Pax6 and Mitf, respectively. From this prototype, all the more complex eye types arose monophyletically. As a mechanism, we propose intercalary evolution of progressively more genes such as lens genes into the eye developmental pathway.



1. http://www.ijdb.ehu.es/web/descarga/paper/041900wg.
2. http://www.cell.com/cell/pdf/S0092-8674(15)01565-2.pdf
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632888/#ref81
4. https://sci-hub.bz/http://www.sciencedirect.com/science/article/pii/030441659290162N?via%3Dihub
5. http://book.bionumbers.org/how-many-rhodopsin-molecules-are-in-a-rod-cell/
6. https://www.sciencedirect.com/science/article/pii/030441659290162N

https://www.nature.com/scitable/topicpage/volvox-chlamydomonas-and-the-evolution-of-multicellularity-14433403



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What was its initial structure and function? Gehring and Ikeo have suggested a two-celled proto-eye made up of one photoreceptor cell and one pigment cell (Gehring and Ikeo, 1999), resembling the two-celled eyes that exist in today’s primary ciliary larvae such as the polychaete trochophore (Fig. 1A) (Arendt et al., 2002) 13

Evolutionary Origin of opsins 2
It is clear that opsins belong to the family-A (or rhodopsin-like superfamily) G-protein-coupled receptors (GPCRs) . Rhodopsin is the best characterized GPCR to date, and it is used as a template to understand other GPCRs. 4
Opsins, and their major divisions  arose very early in metazoan evolution. In this article the term 'opsin' will refer only to 'Type 2 animal opsins', and not to the 'Type 1 microbial opsins' of bacteria or the 'channelrhodopsins' of algae, both of which are unrelated and appear to have arisen by convergent evolution. [url=http://retina.umh.es/webvision/Evolution.%20PART%20I.html#Ciliary photoreceptors in the eyes of extant chordates]5[/url]

Numerous prokaryotic TM proteins probably originated by duplication of their TM domains, evidenced by nonrandom sequence similarity between domains within proteins.

This section moves forward in time from the parental gene content of the immediate ancestral genome (greatly facilitated by the new Trichoplax and Monosiga assemblies) that gave rise to the first opsin via gene duplication and neofunctionalization of one copy to photoreception. 3

Evolution of opsins:
Although parapinopsin ( Any of a group of opsins in the parapineal gland of some fish )  has an amino acid sequence similar to those of vertebrate visual pigments, it has the molecular properties of a bistable pigment, similar to
invertebrate visual pigments (Gq-coupled visual opsin) and Opn3 (encephalopsin)/ TMT-opsin-based pigments. These observations indicate that parapinopsin is one of the key pigments for understanding the molecular evolution of vertebrate visual pigments. Parapinopsin has glutamic acid residues at both positions 113 and 181, similar therefore to vertebrate visual pigments. However, mutational analyses have revealed that Glu181 is the functional counterion residue, as found for invertebrate rhodopsins. Therefore, this suggests that the molecular properties of photoproducts, namely photoregeneration (bistability) and bleaching, may relate to counterion position and that vertebrate visual pigments having bleaching property might have evolved from an ancestral vertebrate bistable pigment similar to parapinopsin. 23

This might be not that easy. In order for the transition to work, all the proteins and enzymes, and all metabolic steps of the visual cycle would have to be set up and in place, fully working. 

If the first opsin was similar to a c-opsin, which some opsin phylogenies suggest, then there would have been an immediate need to evolve a reconversion mechanism, and the evolution of RGR/Go opsins and r-opsins may have been evolutionary responses to this need 22

Now, look THAT tattered guesswork explanation !! The question is a major one and demands a detailed answer. But the authors think that a single line based on " may have " settles the issue. May not !!  These are the questions that decide if an explanatory model makes sense. Unfortunately, Nilssen simply thinks that a short-lined sentence based on " may have" will be enough to draw the attention to other issues. If the need was immediate, and the required proteins were not present, the mechanism simply would not work !! It's either everything or nothing issue. Did opsins using bistable pigments ( which they use still today, and it works just fine ) have  suddenly an urgent need and goal to create a new way of how to do things, and rather than using a bistable retinal, it had to begin using one that bleaches, and so, requiring a complex recycling process ? and so -  create the recycling pathway, and by trial and error during millions of years, somehow, somewhere, in a bit mysterious manner, evolution figured out by gene duplication to come up with a whole set of instructional information how to make the required proteins, and interconnect them together? resulting in a highly complex, regulated, orchestrated new reconversion mechanism - and btw. since an important part requires Retinal pigment epithelium (RPE) cells, which is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells, did evolution of these cells come together in the package ?!! The problem is, the pathway would be unable to recycle all-trans retinal molecules unless the pathway was fully set up and working !! 

Type I and Type II opsins
Opsins comprise two protein families, called type I and type II opsins, with detailed functional similarities, but whose homology is often doubted. Both opsin classes are seven-transmembrane (7-TM) proteins that bind to a lightreactive chromophore to mediate a diversity of responses to light. In both families, the chromophore (retinal) binds to the seventh TM domain via a Schiff base linkage to a lysine amino acid.  Two major classes of opsins are defined and differentiated based on primary protein sequence, chromophore chemistry, and signal transduction mechanisms. Several lines of evidence indicate that the two opsin classes evolved separately, illustrating an amazing case of convergent evolution.

Type I opsins 
are present in bacteria and algae, in both prokaryotic and eukaryotic microbes, functioning as light-driven proton pumps, sensory receptors, and in various other unknown functions. They have varied function, including bacterial photosynthesis (bacteriorhodopsin), which is mediated by pumping protons into the cell, and phototaxis (channelrhodopsin), which is mediated by depolarizing the cell membrane.and are referred to by various names, including 

bacteriorhodopsin 
bacterial sensory rhodopsins
channelrhodopsin 
halorhodopsin 
proteorhodopsin

Type II opsins 
are known only in eumetazoan animals and are involved in the regeneration of converted chromophores and the photosensitive elements of visual perception and circadian rhythms . For two reasons, we herein seek to test a hypothesis on the origins of type II (animal) opsins. First, if type I and type II opsins are homologous, they should share the same origination event. As such, our test of type II origins has implications for homology or nonhomology of opsin types. Second, although an origin by internal domain duplication is supported for some type I opsins, no one has investigated a similar hypothesis for type II opsins. 

Through our experiments, we found no evidence that type II opsins originated by internal domain duplication, consistent with previous claims that type I and type II opsins are not homologous. These results are consistent with multiple other lines of evidence that type I and type II opsins represent an amazing convergence. They are present in eumetazoans (animals not including sponges), but are unknown from sponges or any non-animals. Because opsins are known from cnidarians and bilaterian animals (animals with bilateral symmetry, including humans, flies, and earthworms), Type II opsins are inferred to have been present in their common ancestor, which lived about 600 million years ago. Type II opsins have varied function, including 

-phototransduction and vision
-circadian rhythm entrainment
-mediating papillary light reflex (pupil constriction)
-photoisomerization (recycling the chromophore)


Despite their functional similarity and despite both being 7-transmembrane proteins,






 Multiple lines of evidence indicate that Type I and Type II opsins evolved independently. 

First, the primary amino acid sequences of Type I and Type II opsins are no more similar than expected by chance. For example, try to align a Type I (say bacteriorhodopsin) and Type II opsin together. 
Second, The orientation of the transmembrane domains differs between the major groups. We now have crystal structure data for both Type I and Type II opsins, and the arrangements of the parts of the protein that are stuck in the cell membrane are quite different, inconsistent with a single origin of opsins (unless this changed a lot during evolution, which is not impossible).
Third, the major opsin groups differ in chromophore chemistry. Prior to light activation, the chromophore of Type I opsins is an all-trans isomer. Light activation then involves isomerization of the chromophore to 13-cis retinal. In contrast, prior to light activation, the chromophore of type II opsins is 11-cis retinal. Light activation of Type II opsins involves isomerization to all-trans retinal.
Fourth, Type II opsins belong to the larger protein family called G-protein coupled receptors (GPCRs), which transmit varied signals from outside to inside cells by activating GTPase proteins, which in turn signal to second messengers that affect the state of the cell in various ways. Type I opsins do not activate G-proteins. Furthermore, Type II opsins are more closely related to non-opsin, light insenstive GPCR’s than they are to Type I opsins. So even if there is some very, very distant and *undetectable* common origin of Type I and Type II opsins, chromophore binding likely evolved twice. Since chromophore binding is what allows photosensitivity, it is the crux of being an opsin, and the realization that Type II opsins are closer to non-opsin GPCR's than Type I opsins is strong support for two separate origins.



While lower organisms utilize the microbial rhodopsin family for light–energy conversion and intracellular signaling, animals use the photosensory functions of a different family of rhodopsins (animal rhodopsin), a specialized subset of G protein-coupled receptors (GPCRs). Microbial and animal rhodopsins share a common architecture of seven-transmembrane α-helices with the N- and C-terminus facing outwards from and inside of the cell, respectively, but have almost no sequence homology and differ largely in their functions . 

Opsins can be divided and limited to particular opsin subgroups (for instance, from vertebrates or arthropods)

Four major monophyletic groups of opsins have been previously defined: those found in 

- ciliary photoreceptors (‘c-opsins’)
- rhabdomeric photoreceptors (‘r-opsins’)
- cnidarians opsins (‘Cnidops’) and a mixed group consisting of 
- retinal G-protein coupled receptors (RGR), peropsins and neuropsins






A route for the evolution of photoreceptor cell types and different forms of eyes. 
a, The cnidarian–bilaterian ancestor had photoreceptors that expressed c-opsin and PAXB. 
b, Rhabdomeric photoreceptors, r-opsins and PAX6 evolved in ancestral-stem bilaterians, after the split between the cnidarian and bilaterian lineages. 
c, The last common ancestor of all bilaterians (Urbilateria) probably had two types of light-sensing organ: a prototypical eye and a brain photo-clock, which are both found in the annelid Platynereis dumerilii
d, The photoreceptor types established in the Urbilateria were then incorporated in different ways in the parallel evolution of different eyes in various phyla. Rhabdomeric photoreceptors were the foundation for the evolution of compound and camera-type eyes in arthropods and molluscs, respectively. Both types of photoreceptor were incorporated into the vertebrate eye, with ciliary receptors carrying out phototransduction and rhabdomeric receptors being transformed into ganglion cells and functioning in image processing. Pigment cells are not shown. 
e, The ciliary camera-type eyes of box jellyfish are also proposed to have evolved in parallel in the cnidarian lineage. 
f, Cladogram depicting the evolutionary relationships of the taxa shown in a–e. 



Fig. 3. Two lines of signal transductions in the animal kingdom. 
Animal phototransduction cascades are evolutionary and functionally classified into two groups, cyclic nucleotide signaling in ciliary-type photoreceptor cell with disk membranes of modified cilia (left) and phosphoinositol signaling in rhabdomeric-type photoreceptor cell with apical microvilli (right). Representative animal photoreceptor cells are indicated. PDE, phosphodiesterase; GC, guanylyl cyclase; AC, adenylyl cyclase; CNG channel, cyclic nucleotide gated channel; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; DG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; TRP channel, transient receptor potential channel; TRPL channel, transient receptor potential-like channel. 23


Biologists have historically inferred that eyes had evolved independently dozens of times . But deciding where homology ends and novelty begins is not always straightforward, particularly for structures that lack a fossil record, such as eyes. 15  In all light-sensing organs in animals, the ability to detect light depends on a cascade involving opsin proteins. This observation led to the view that all modern variations of light sensing in bilaterians can be traced to the existence of photosensitive cells in a common ancestor with PAX6 and other transcription factors at the top of a genetic regulatory pathway leading to opsin production. This is a textbook example of deep homology1,2: morphologically disparate organs whose formation (and evolution) depends on homologous genetic regulatory circuits.  The photoreceptors of different taxa are of two main types, rhabdomeric and ciliary (Fig. 1b), which have distinct phototransduction signalling cascades. The eyes of insects and other invertebrates rely on rhabdomeric photoreceptors and a phospholipase-C-based cascade, whereas vertebrates use ciliary photoreceptors and a phosphodiesterase-based cascade. How can the fact that similar camera-type eyes of vertebrates and squids rely on different kinds of photoreceptor and transduction process be explained, other than by asserting completely separate origins? 15





Rhabdomeric and ciliary photoreceptors and their homologies. 
Conservation of cell polarity and topology between Drosophila rhabdomeric photoreceptor (left) and vertebrate rod photoreceptor (right). The center image pair, in top and side view, depicts an ancestral (or an immature) photoreceptor, which can evolve (or develop) into either of the two illustrated final forms simply by different modes of expansion of the apical membrane (pink); that apical membrane is separated from the baso-lateral membrane (yellow) by a zonula adherens (ZA, brown). r, rhabdomere; s, fly stalk; os, outer segment; cc, connecting cilium; is, inner segment; ELM, external limiting membrane; N, nucleus.


Two types of ion channel delineate different modes of phototransduction known in animals. 

Ciliary phototransduction cascade

Cyclic nucleotide–gated (CNG)  ion channels function in a ciliary phototransduction cascade, whereby CNG modulation effects a hyperpolarizing potential in the photoreceptor cell's response to light.

Cyclic nucleotide–gated ion channels or CNG channels are ion channels that function in response to the binding of cyclic nucleotide (cNMP). 

cyclic nucleotide (cNMP) is a single-phosphate nucleotide with a cyclic bond arrangement between the sugar and phosphate groups. Like other nucleotides, cyclic nucleotides are composed of three functional groups: a sugar, a nitrogenous base, and a single phosphate group. As can be seen in the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) images, the 'cyclic' portion consists of two bonds between the phosphate group and the 3' and 5' hydroxyl groups of the sugar, very often a ribose.




Cyclic adenosine monophosphate. The cyclic portion refers to the two single bonds between the phosphate group and the ribose


Cyclic nucleotides are produced from the generic reaction NTP → cNMP + PPi, where N represents a nitrogenous base. The reaction is catalyzed by specific nucleotidyl cyclases, such that production of cAMP is catalyzed by adenylyl cyclase and production of cGMP is catalyzed by guanylyl cyclase    Adenylyl cyclase has been found in both a transmembrane and cytosolic form, representing distinct protein classes and different sources of cAMP.


CNG channels are nonselective cation channels that are found in the membranes of various tissue and cell types, and are significant in sensory transduction as well as cellular development. Their function can be the result of a combination of the binding of cyclic nucleotides (cGMP and cAMP) and either a depolarization or a hyperpolarization event. Initially discovered in the cells that make up the retina of the eye, CNG channels have been found in many different cell types across both the animal and the plant kingdoms. CNG channels have a very complex structure with various subunits and domains that play a critical role in their function. CNG channels are significant in the function of various sensory pathways including vision and olfaction, as well as in other key cellular functions such as hormone release and chemotaxis. CNG channels have also been found to exist in prokaryotes, including a large number of spirochaeta, though their precise role in bacterial physiology remains unknown.

Rhabdomeric pathway

Conversely, canonical transient receptor potential (TRPC) ion channels, members of a larger gene family of TRP channels , function in a rhabdomeric pathway where activation leads to a depolarizing cell-physiological response to light

Both CNG- and TRPC-modulated pathways are initiated by class-specific opsin paralogues (c-opsin and r-opsin, respectively), which are both present in protostomes and deuterostomes, and therefore predate the origin of bilaterian animals. Despite the centrality of these signalling pathways to vision and other photosensitivity phenotypes, an understanding of the origins of these cascades in animal evolution has evaded biologists, largely because accumulation of data from non-bilaterian animals like Cnidaria has lagged far behind the availability of data from bilaterians—especially flies, molluscs and vertebrates. Despite the centrality of these signalling pathways to vision and other photosensitivity phenotypes, an understanding of the origins of these cascades in animal evolution has evaded biologists


In chemistry isomerization (also isomerisation) is the process by which one molecule is transformed into another molecule which has exactly the same atoms, but the atoms have a different arrangement e.g. A-B-C → B-A-C  4

Upon illumination, 11-cis retinal isomerizes into the all-trans configuration and initiates protein-protein interactions (not ion flux) that trigger the visual phototransduction second messenger cascade.

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 4


Based on sequence homology, the opsin family can be categorized into six subfamilies, namely 

the vertebrate opsin/encephalopsin subfamily, 
the Go opsin subfamily, 
the recently characterized Gs opsin subfamily, 
the invertebrate Gq opsin subfamily, 
the photoisomerase subfamily and 
the neuropsin subfamily. 

Opsin diversity has been further subdivided to form up to 22 subgroups found in the vertebrates alone


A molecular phylogenetic tree of the opsin family. 20
members of opsin family are divided into seven subfamilies, whose names are given on the right of the tree. Common names of species shown: Anopheles, mosquito; Branchiostoma, amphioxus; Ciona, ascidian; Drosophila, fruit fly; Patinopecten, scallop; Platynereis, polychaete annelid worm; Procambarus, crayfish; Schistosoma, blood fluke; Todarodes, squid. Abbreviations: LW, long-wavelength-sensitive opsin; SW, short-wavelength-sensitive opsin; MW, middle-wavelength-sensitive opsin; Rh, rhodopsin; RGR, retinal G-protein-coupled receptor. Other abbreviations are protein names; where only a color is given for a protein name, it refers to a cone opsin that detects that color.

The visual and non-visual opsin subfamily contains vertebrate visual and non-visual opsins. 

The visual opsins 
can be further subdivided into cone opsins and rhodopsin, which have distinct molecular properties arising from differences in the residues at positions 122 and 189 of the amino-acid sequence.

The cone opsins can be further divided into four subgroups, which correspond well with their absorption spectra: 

- long-wavelength opsins (LW or red), 
- short-wavelength opsins SW1 or UV/violet 
- SW2  blue 
- middle-wavelength opsins (MW or green ) 


Schematic representation of the phylogenic relationship of opsins. 
Opsins belong to the family-A GPCRs, and they can be roughly subdivided into ciliary opsinsrhadbomeric opsins and photoisomerases. The ciliary opsins are characterized by their expression in ciliary photoreceptor cells and cyclic nucleotide signalling cascade. On the other hand, rhabdomeric opsins are expressed in rhabdomeric photoreceptor cells and have phosphoinositol signalling cascade. Finally, photoisomerases comprises proven and putative stereospecific photoisomerases.




Figure 1.

The origin of vertebrates, over a time-scale from roughly 700 to 400 million years ago (Mya), with timings of the branchings taken from a reconciliation of fossil and molecular data by Erwin et al (2011). The red curve indicates our direct ancestors, beginning with early metazoans, and the numbers along the curve denote major branchings that are significant to the evolution of the vertebrate eye. #1, Cnidarians branched off around 700 Mya, and since then our ancestors have been bilaterally symmetric (bilaterians). #2, Protostomes branched off around 665 Mya, and since then our ancestors have been termed deuterostomes. #3, #4, By the time that cephalochordates and then tunicates diverged (around 650 and 600 Mya), our ancestors possessed a notochord, and are referred to as chordates. #5, By the time that the ancestors of lampreys diverged around 500 Mya, they and our own ancestors were vertebrates. It has recently been confirmed that the '2R' two rounds of whole-genome duplication occurred prior to that divergence of ancestral lampreys. Subsequently (by around 420 Mya) our own ancestors evolved jaws and hence became jawed vertebrates (gnathostomes). Black curves indicate taxa that are not considered in any detail in this article; dashed black curves indicate extinct taxa of potential interest. See text for details. Source of illustrations: Haikouella, Yunnanazoon and Haikouichthys from Chen (21); remainder from Lamb et al (3), where original sources are given.

Origin of opsins  21

A-1) The forerunner of the first opsin arose through duplication of a GPCR in an ancient metazoan, at a time prior to the divergence of the amoeba-like placozoans.

A-2) That forerunner protein did not possess the retinal-binding lysine ('K296') in the seventh transmembrane helix (30); this suggests that retinaldehyde ligand occupied the internal cavity by means of non-covalent binding, as for ligands in conventional GPCRs, and in Figure 2B this pre-opsin is termed a 'retinaldehyde receptor'. The placozoan Trichoplax has a homolog of opsin (dubbed placopsin by Feuda et al, 2012), that likewise is devoid of the retinal-binding lysine residue.

A-3) Acquisition of an appropriately situated lysine residue within the seventh transmembrane segment of that receptor allowed the retinaldehyde ligand to bind covalently. Initially, the Schiff base bond is likely to have been unprotonated, so that the molecule would have absorbed in the UV. Acquisition of an appropriately located negatively charged residue (e.g. E181) permitted the bond to be protonated, thereby creating the ancestral opsin, and enabling the absorption peak to be shifted into the 'visible' spectrum.

A-4) As for most opsins (though not for vertebrate visual opsins), the activated metarhodopsin state of this opsin was thermally stable and could undergo photoreversal to the rhodopsin state. Hence this protein probably did not require a source of 11-cis retinal and could instead utilize all-trans retinal perfectly well.

A-5) Subsequently, two duplications of that earliest opsin occurred, during the relatively short interval between the divergence of placozoa and the divergence of cnidarians from bilaterians. Thus, all of the duplications indicated in Figure 2B took place shortly prior to the first of the numbered branchings shown in Figure 1 (i.e. prior to #1).

Hypothesized association between opsin type and membrane type . A contributory factor in the co-evolution of opsin classes and photoreceptor classes may have been a preferential association of the different opsins with different regions of membrane, as indicated in Figure 2B. Accordingly, the hypothetical scenario for the early evolution of opsins is extended as follows:

A-6) The two variants of opsin that emerged after the first duplication event may have trafficked preferentially to the membrane of sub-cellular organelles and to surface membrane. Those variants would have given rise to the RGR- division and the C-/R- division, respectively, of modern opsins.

A-7) Following the duplication event that created the distinction between C- and R-opsins, these two variants trafficked to ciliary and microvillar membrane, respectively. In Figure 2B this duplication is shown as having occurred subsequent to the duplication mentioned in the previous point, but at present one cannot reliably distinguish the order in which this pair of duplication events occurred.

A-8 ) Subsequently, cells expressing the C- and R-opsin classes became distinct from each other, through a process termed 'division of labor' (5, 33), leading to (a) ciliary photoreceptors that possessed C-opsins and (b) microvillar photoreceptors that possessed R-opsins; see next Section. The third variant of opsin, RGR-opsin, tended to be expressed in the membranes of intracellular organelles, possibly as an additional opsin in the first two classes of photoreceptors.

A-9) Later in evolution, further division of labor occurred, so that (for example) RGR-opsin could be expressed in separate cells. This would explain how it is possible, on the one hand, for squid photoreceptors to contain an R-opsin in their microvillar membranes as well as retinochrome (an RGR-opsin) in their intracellular organelles, and, on the other hand, for vertebrate cones and rods to contain only a C-opsin in their outer segments whereas RPE cells contain only RGR-opsin in their endoplasmic reticulum.



Figure 2.

Origin of opsins, and their possible association with membrane type. 
A, Opsin phylogeny. Cnidarians have orthologs of each bilaterian opsin subfamily; i.e. the C-, R-, and RGR/Go-opsin subfamilies. Numbers indicate support values (Bayesian PPs) for key nodes. From Feuda et al (30). 
B, Hypothesized duplications of ancestral opsin and its precursors, and suggested association with membrane type. An ancient GPCR (related to extant vertebrate melatonin receptors) duplicated, and its ligand became retinaldehyde, which bound non-covalently; this is denoted as 'Retinaldehyde receptor'. After the divergence of the amoeba-like placozoans (~711 Mya), this GPCR evolved a lysine residue in its seventh transmembrane segment and a negatively charged residue (counterion) so that retinaldehyde bound covalently via a protonated Schiff base linkage; this form is denoted 'Ancestral opsin'. Within a relatively short interval (prior to the divergence of cnidarians, ~700 Mya), this opsin duplicated twice, giving rise to three major families of opsins: C-opsins, R-opsins, and RGR/Go-opsins. It is proposed that these three opsins preferentially associated with ciliary membrane, microvillar membrane, and the membranes of intracellular organelles, respectively. Note that all these events occurred just prior to the starting point of Figure 1.

The evolution of phototransduction from an ancestral cyclic nucleotide-gated pathway 9
The evolutionary histories of complex traits are complicated because such traits are comprised of multiple integrated and interacting components, which may have different individual histories. 

What is almost completely neglected is the fact that these components, individually, on their own, do exercise in most cases no function. This point alone makes evolution impossible. Nobody in its sane mind would invent a piston by its own, without considering the complete engine and motor block and its end function. 

Phylogenetic studies of complex trait evolution often do not take this into account, instead focusing only on the history of whole, integrated traits; for example, mapping eyes as simply present or absent through history. Using the biochemistry of animal vision as a model,  investigating the individual components of complex systems can aid in elucidating both the origins and diversification of such systems. Opsin-based phototransduction underlies all visual phenotypes in animals, using complex protein cascades that translate light information into changes in cyclic nucleotide gated (CNG) or canonical transient receptor potential (TRPC) ion-channel activity. Here we show that CNG ion channels play a role in cnidarian phototransduction. Transcripts of a CNG ion channel co-localize with opsin in specific cell types of the eyeless cnidarian Hydra magnipapillata. Further, the CNG inhibitor cis-diltiazem ablates a stereotypical photoresponse in the hydra. 

Our findings in the Cnidaria, the only non-bilaterian lineage to possess functional opsins, allow us to trace the history of CNG-based photosensitivity to the very origin of animal phototransduction. Our general analytical approach, based on explicit phylogenetic analysis of individual components, contrasts the deep evolutionary history of CNG-based phototransduction, today used in vertebrate vision, with the more recent assembly of TRPC-based systems that are common to protostome (e.g. fly and mollusc) vision.

Animal phototransduction cascades are prime examples of complex traits, with components that have been well characterized on functional, biochemical and genetic levels. Despite this progress, the evolutionary origins of these multi-component cascades remain little understood and have been the subject of both longstanding puzzlement among evolutionary biologists (Darwin 1859, 1987) and cynicism among others (Behe 1996).



Figure 2.
Ancestral state reconstruction supports the hypothesis that CNG ion channels functioned in the ancestral phototransduction cascade. A phylogeny for animal opsins represents the evolutionary history of the phototransduction cascades. Outgroups (grey) were selected based on a previous study of GPCR evolution (Fredriksson et al. 2003). Functional relationships between specific opsin sequences and their ion channel types were coded based on new data reported here and those abstracted from the literature (orange, CNG; blue, TRPC; black, no phototransduction as per photoisomerases).  Additional sequences (white) mined from public databases (see §2). Reconstruction of ancestral states using character mapping reveals, with a strong proportion of likelihood (pie chart), that the ancestral animal phototransduction cascade used a CNG ion channel (orange; proportion of likelihood = 0.99). The node joining the rhabdomeric opsin is also reconstructed, with a high proportion of likelihood, to have used a TRPC ion channel (blue; proportion of likelihood = 0.99). Additional analyses using Bayesian methodology strongly support these findings. Outgroup rooted ML tree is shown (see electronic supplementary material, figure S5 for support indices). Posterior probabilities (PP) of reconstructed states from Bayesian analyses integrate over all topologies sampled from the posterior (electronic supplementary material, figure S6) and allow multiple character state transitions to occur along branches.

The most primitive extant metazoans with eyes are the cnidarians (corals, sea anemones, jellyfish, and hydroids). All photoreceptors of adult jellyfish that have been studied are ciliary with opsins distantly related to the c-opsins of vertebrates 11



Phylogenetic tree of metazoans showing only animal groups or species discussed in this review, with photoreceptor types in principal eyes illustrated as ciliary (red) or microvillar (blue)
No attempt has been made to specify relationships among principal groups of bilaterians, since these remain controversial. Cnidarian embryos may have microvillar photoreceptors (see text), and it is likely that both photore-ceptor types were present very early in the evolution of metazoans. Mammals and other vertebrates have ciliary photoreceptors (rods and cones) and do not have photoreceptors with microvilli, but they may use a transduction cascade similar to the one used by microvillar photoreceptors in the intrinsically light-sensitive ganglion cells [16]. Phylogenetic tree is based upon [54,94,99]. Drawings of photoreceptors are from [3,5,56,61,95,100103].




Examining the histories of their individual components can illuminate the origination of complex traits. 
(a) Illustrated is a traditional view of animal phylogeny (Philippe et al. 2009) with the presence (grey) and absence (white) of opsin-based phototransduction mapped onto the tree using parsimony. This approach illustrates that cnidarians, including Hydra magnipapillata, are in a key phylogenetic position to inform our view of the origins of this complex trait. 
(b) Analysing the individual components of phototransduction in a phylogenetic context yields deeper insights into its origin and evolution. Round-cornered rectangles represent phototransduction (grey) or other G-protein-coupled receptor (GPCR) pathways (white), with individual components inside. Our results indicate that CNG ion channels are involved in hydra phototransduction (figure 1) as well as the ancestral phototransduction cascade (figure 2). Because CNG is present outside eumetazoan animals and is involved in non-opsin GPCR pathways (Stumpf et al. 2009), we infer that—like G-proteins and other phototransduction components (Suga et al. 1999Plachetzki et al. 2007)—CNG ion channels predate phototransduction. Therefore, the origin of phototransduction may have involved the gain of light sensitivity in an ancient CNG ( cyclic nucleotide-gated (CNG) ion channel ) –GPCR ( G-protein-coupled receptors (GPCRs) ) pathway. Our results also indicate that TRPC-based rhabdomeric phototransduction originated by changing multiple components. Protostomes and deuterostomes are illustrated to have both ciliary and rhabdomeric pathways (Arendt 2003), with the pathway dominant in vision positioned on top. Dashed lines indicate major clades in metazoan evolution. Additional abbreviations: c-opsin, ciliary opsin; r-opsin, rhabdomeric opsin; I, intermediary molecules; Ic, ciliary intermediaries such as phosphodiesterase (PDE) and guanylate cyclase (GC); Ir, rhabdomeric intermediaries such as phospholipase C (PLC), DAG (diacylglycerol) and PIP2 (phosphatidylinositol-4,5-biphosphate).

We infer that the CNG ion channel component of the ancestral phototransduction pathway was present prior to the origin of animal opsins. In addition, earlier studies showed that G-proteins and other components of animal phototransduction cascades diversified prior to Metazoa .


The origin of phototransduction may have involved the gain of light sensitivity in an ancient CNG ( cyclic nucleotide-gated  ion channel ) –GPCR ( G-protein-coupled receptors (GPCRs) ) pathway.


16



The apparent lack of transitional forms that have been preserved during the course of vertebrate eye evolution has provided perennial fodder for ‘creationists’. But, as Charles Darwin (1859) explained,

“if numerous gradations from a perfect and complex eye to one very imperfect and simple, each grade being useful to its possessor, can be shown to exist… and if any variation or modification in the organ be ever useful to an animal under changing conditions of life, then the difficulty of believing that a perfect and complex eye could be formed by natural selection, though insuperable by our imagination, can hardly be considered real”.

One of the goals of this article is to document evidence for such gradations in the photoreceptors, in the phototransduction cascade, and in the retina, during the course of chordate and vertebrate evolution. A second major aim is to construct a set of ‘scenarios’ for the long sequence of events that contributed; in this regard the term ‘scenario’ is used in its dictionary sense of ‘a postulated sequence or development of events’. 7

Opsin proteins are essential molecules in mediating the ability of animals to detect and use light for diverse biological functions. Therefore, understanding the evolutionary history of opsins is key to understanding the evolution of light detection and photoreception in animals. 12   The discovery of consistent differences in photoreceptor structure, primary opsin gene sequence and signal transduction components between vertebrate and protostome visual pigments has led to the conceptual division of opsin diversity into two basic classes: the ‘r-type’ and ‘c-type’ opsins




The four processes 
a, molecular components; 
b, cell structures; 
c, cell types; 
d, organ shape) involved in eye evolution. 


The processes overlap in time, but are initiated in the ascending order. 14

Evolution of Visual and Non-visual Pigments page 111

The evolutionary explanation of how the molecules that confer vision evolved:

All opsins, invertebrate and vertebrate, derive from a common ancestral G-protein-coupled receptor . 17 The opsins of animals are evolutionarily and functionally quite distinct from two other photoproteins with similar names: the bacteriorhodopsins of halobacters and the channelrhodopsins found in green algae (recently famous for their use in optogenetics). As mentioned, animal opsins in all their diversity are GPCRs (although not all actually activate a G-protein) and all descend from a common ancestor.

Opsins arose at about the time of the appearance of the earliest eumetazoans, today represented by cnidarians and ctenophores. Of earlier animals, sponges (Porifera) detect and respond to light, but no opsins have been identified in sponges. Instead, their photoreception—when present—is apparently based on a cryptochrome photopigment

It can be reasonably argued that everything to do with photosensing and vision was invented before the radiation of the major animal groups.

The molecular components such as opsins and the transduction proteins are subject to variations in the protein sequence, introduced by random mutations. Selection acts on sequence changes, and because the vast majority of changes are neutral or detrimental, the small choice available to selection will make the direction of sequence evolution depend largely on the random nature of mutations, and the process generates discrete rather than continuous change. Genes may have more than one role, and this gene sharing has important consequences for change. Changes in developmental genes are ultimately responsible for the coordinated expression of proteins that form the cell structures. The gradual and quantitative nature of such modifications is typically the result of interactions between many genes (Futuyma 1986), and as such, the randomness of mutations would be expected to have only minor impact on the evolution of these features. Evolution of cell types, which can most often be described as cell specialization, generally requires a growing number of separate cell types. Cell-type duplication followed by segregation is thought to be a central mechanism in the evolution of metazoan complexity. It is believed that many of the specialized cells in animal retinas have originated from ancestral cells with multiple functions, much like changing from a one-man band to a large orchestra. Such principles are believed to be responsible for the evolution of different receptor cell types, pigment cells and interneurons from ancestral receptor cells

That's the standard claim. And superficial explanation in order to keep the darwinian explanation alive. But will it withstand scrutiny and close examination ?

Opsins bind a light-sensitive chromophore and signal when the chromophore has been altered by light. In opsins, the chromophore is a vitamin-A derivative, most commonly vitamin-A aldehyde, also known as retinal, which responds to light by changing from an 11-cis to an all-trans conformation. Opsins are thus still behaving as chemoreceptor proteins, but for a specific light-induced chemical stimulus, and it is possible that this reflects a chemoreceptor origin of animal opsins.

There are different classes of opsins that behave in different ways when the chromophore is isomerized. The c-opsins (originally known from vertebrate rods and cones) release the chromophore after it has been converted to the all-trans isoform (Lamb & Pugh 2004), whereas the r-opsins keep both the 11-cis and the all-trans isoforms firmly bound (r-opsins are known from the photoreceptors of invertebrate eyes and also represented by melanopsins in vertebrates). In the r-opsins, the chromophore can be converted back to the 11-cis form by the absorption of yet another photon, and this photoregeneration serves to replenish sensitive photopigment (Hamdorf 1977). The c-opsins cannot themselves regenerate the chromophore, and a separate enzymatic system is required for this purpose.

Most invertebrate eyes are based on rhabdomeric receptor cells, whereas vertebrate and cnidarian eyes are based on ciliary receptor cells. The distinction between ciliary and rhabdomeric receptor cells is emphasized by the fact that they strictly employ different classes of opsin, c-opsin and r-opsin, respectively, and as a consequence, they also use different transduction cascades 

Presently, it seems that the structure and design of eyes are so different between animal phyla that, for the most part, eye evolution has proceeded independently in different animal groups.  A prerequisite for visual photoreception is that it provides information about the direction of light, either by partially shielded photoreceptors and scanning body movements 

Light sensitivity

Detection of light by animal opsins implies that 11-cis chromophore is consumed. To maintain sensitivity, active chromophores must be replenished at a rate comparable to consumption. Direct sunlight corresponds to 10 million photons of visible light per second reaching each square micrometre of the earth's surface, and this consumes the 11-cis chromophore at an extreme rate in exposed photoreceptor cells. However, spontaneous (thermal) regeneration from an all-trans to an 11-cis retinal only occurs at very low rate (Deng et al. 1991). Using light also to flip the chromophore back from all-trans to 11-cis configuration solves the problem, but for free chromophore, this requires ultraviolet (UV) radiation that is absent even at moderate depths in the sea. The photoisomerases, which are non-sensory opsins, have the property of regenerating the chromophore in the presence of visible light, and as such, they are complementary to c-opsins, which lack this ability.

That means, C-opsins DEPEND on photosisomerases for regeneration. Interdependence is evidence of design, since one would not function without the other. They had to emerge together. What if not intelligence alone is able to coordinate it ? 

The ancestral opsin is unlikely to have been a photoisomerase because it is hard to see a need for such an enzyme if there is no receptor protein producing its substrate.

It's also hard to see why such an enzyme would have evolved unless it knew , once fully evolved, the task it would fullfill. 

The only remaining scenario is that an ancestral opsin had c-opsin properties and that the first gene duplication led to the evolution of a photoisomerase that would form an efficient functional pair together with the ancestral opsin.

Class I: Nondirectional photoreception

By just monitoring the ambient light intensity, it is possible to get information for a number of important behaviors such as directly triggering or turning off behaviors in relation to the time of day or indirectly by providing input to a circadian clock. Aquatic animals can also use the ambient intensity as a depth gauge to control their vertical position in the water column. Burrowing animals may use nondirectional photoreception to trigger appropriate behaviors when the animal breaks the substrate surface and enters the lit world. Shadow detection can inform animals when they move into or out of shadows or when a shadow moves in on the animal. Warning for harmful levels of UV radiation is another task that is essential for many animals.

Molecular, developmental, and morphological studies have revealed some common ground in the eyes of virtually all multicellular animals. 6 All photoreceptors use a light-sensitive pigment derived from vitamin A, and this pigment is bound to a protein called opsin. Light activates opsin by causing a conformation change in the photopigment, and opsin then binds to a G-protein, a common and versatile molecule used in many signal transduction cascades.  This sequence of events turns light into a chemical signal.

Rhabdomeric photoreceptors

Rhabdomeric photoreceptors are found in the compound eyes of arthropods. They increase their surface area by throwing up their apical surfaces into numerous folds—in some forms, the cell looks like it has had a flat-top crewcut, with a crowning bristle of fine membranous bristles, although the cell itself can have many different shapes in different species.

Signal transduction in rhabdomeric photoreceptors involves activation of phospholipase C (PLC) and the inositol phosphate (IP3) pathway.

Ciliary photoreceptors

The increase in membrane surface area in ciliary photoreceptors, the kind of receptor we vertebrates use, is by modification of the cilium, a process that extends from the cell. The ciliary membrane is expanded and thrown into deep folds, so that the actual receptor region of the cell looks like a stack of discs.

Ciliary photoreceptors use a different signalling pathway, activating a phosphodiesterase (PDE) that changes the concentration of cyclic GMP in the cell. Both the IP3 and the PDE pathways exist in all animals; the difference is in which pathway is used in the different photoreceptors. The diagram below illustrates the two different pathways, and also shows the phylogenetic relationships between their different molecular components (beware of tiny print! Click on the image for a more readable verson).

Chlamydomonas rheinhartii might more strictly be defined as a green algae in the plant kingdom. Phototaxis is essential for both organisms; 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 Chlamydomonasand Euglena. 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. 8

In Chlamydomonas the eyespot apparatus consists of thylakoid membranes with layers of carotenoid rich globules and photoreceptor molecules in the membranes between these globules 
The problem here is that 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. 

Having said that investigation of opsin diversity sheds considerable light on the evolution of life once we get beyond the protist stage.  There are two lines of photoreceptors, those involving animals with photosensitive cilia and those with rhabdomeres. The latter are the Protostomia including the arthropods, whereas the former are the Deuterostomes that include the vertebrates. They have different opsins (R and C) and different mechanisms of converting light signals to nerve impulses; 

- C opsins functioning through a cyclic nucleotide pathway, whereas 
- R opsins use phospholipase C for signal transduction. 

1. https://sci-hub.bz/http://www.nature.com/nature/journal/v482/n7385/abs/nature10870.html
2. https://sci-hub.bz/http://www.nature.com/nature/journal/v482/n7385/full/482318a.html
3. http://www.ntskeptics.org/creationism/evolution/OpeningTheBlackBox.pdf
4. https://en.wikipedia.org/wiki/Isomerization
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483371/
6. http://scienceblogs.com/pharyngula/2006/09/14/rhabdomeric-and-ciliary-eyes/
7. http://www.sciencedirect.com/science/article/pii/S1350946213000402
8. http://www.nature.com/eye/journal/v30/n2/full/eye2015220a.html
9. http://rspb.royalsocietypublishing.org/content/277/1690/1963
10. https://en.wikipedia.org/wiki/Cyclic_nucleotide
11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898276/
12. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223661/
13. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.460.6560&rep=rep1&type=pdf
14. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781862/
15. https://sci-hub.bz/http://www.nature.com/nature/journal/v457/n7231/full/nature07891.html
16. http://onlinelibrary.wiley.com/doi/10.1002/wmts.6/pdf
17. Evolution of Visual and Non-visual Pigments, page 111
18. http://evolutionarynovelty.blogspot.com.br/2008/07/evolutionary-origins-of.html
19. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632888/
20. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1088937/
21. https://www.ncbi.nlm.nih.gov/books/NBK153508/
22. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632888/#ref81
23. http://www.sciencedirect.com/science/article/pii/S000527281300159X#f0010
24. http://www.readcube.com/articles/10.3389/fevo.2017.00023

Further readings:
Could the eye have evolved by natural selection in a geological blink?
https://uncommondescent.com/intelligent-design/could-the-eye-have-evolved-by-natural-selection-in-a-geological-blink/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781865/

Evolution of phototransduction, vertebrate photoreceptors and retina
http://www.sciencedirect.com/science/article/pii/S1350946213000402

Eye evolution and its functional basis
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632888/



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3 The phototransduction pathway - how it works on Thu Oct 12, 2017 7:45 pm

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The  phototransduction pathway - how it works

In vertebrate photoreceptors, photon absorption and visual signaling take place in the outer segment , a sensory cilium tightly packed with stacks of membranous discs containing extremely high densities of visual pigments and other signaling proteins. This morphological arrangement allows photons to be efficiently absorbed as they pass through the outer segment.  The signal from activated visual pigment (rhodopsin in rods or cone opsins in cones) must then be sufficiently amplified to generate an electrical response that overcomes intrinsic noise. Transduction from the light-absorbing visual pigment into an electrical response utilizes a G protein signaling pathway termed the phototransduction cascade, which leads to a decrease in the second messenger cGMP and the closure of cGMP-sensitive cation channels. The resulting hyperpolarization transiently decreases the release of glutamate from the photoreceptor synaptic terminals, signaling the number of absorbed photons to the rest of the visual system. Remarkably, photoreceptors both detect low light levels (single photons in the case of rods) and continue to rapidly and reliably signal changes in light intensity as illuminance increases over 10 orders of magnitude during the course of a typical day.

The light-sensitive properties of rhodopsin, the vertebrate rod photoreceptor pigment, originate from its 11- cis -retinal chromophore. The absorption of light by rhodopsin results in the isomerization of the 11- cis -retinal chromophore to all- trans forming the enzymatically active intermediate, metarhodopsin II, which commences the visual transduction process. Photoisomerization of the 11- cis -retinal chromophore to all- trans renders rhodopsin insensitive to light. To regain sensitivity to light, all- trans -retinal has to be removed and fresh 11- cis -retinal be supplied to regenerate rhodopsin. These reactions take place in the outer segment of the rod photoreceptor, where rhodopsin is densely packed. Removal of all- trans -retinal begins with the hydrolysis of the Schiff base that links it covalently to opsin, the protein part of rhodopsin. All- trans -retinal is then released, leaving behind opsin, which can combine with fresh 11- cis -retinal to regenerate rhodopsin. The removal of all- trans -retinal is completed through reduction to all-trans - retinol in a reaction catalyzed by the enzyme retinol dehydrogenase  and using NADPH as a cofactor. Subsequently, in a process facilitated by the interphotoreceptor retinoid-binding protein (IRBP), all- trans -retinol leaves the rod outer segment and is transferred to the adjacent cells of the retinal pigment epithelium (RPE). There, all- trans -retinol enters a series of reactions that culminate in the generation of 11- cis -retinal. Another source of all- trans -retinol is the choroidal circulation, which delivers it bound on retinol-binding protein (RBP) to the basal side of the RPE cells. Inside the RPE cells, all-trans - retinol is esterified by lecithin/ retinol acyltransferase (LRAT) to form retinyl esters, which are the substrate used by the RPE65 protein to make 11- cis -retinol. Retinol dehydrogenase RDH5 oxidizes 11- cis -retinol to 11- cis -retinal, which is then transported from the RPE cells to rod outer segments by IRBP.

Because of its importance for maintaining light sensitivity, rhodopsin regeneration has been and is being studied extensively at the molecular, cellular, and whole organism levels. Additional importance of the reactions involved in rhodopsin regeneration stems from the involvement of the highly reactive allylic aldehydes 11- cis and all- trans -retinal. The cytotoxic properties of all-trans -retinal have been extensively documented and studied; on the basis of its structure, the toxic effects of 11- cis -retinal would be expected to be similar. The cytotoxic potential of 11- cis and all-trans - retinal extends even further, as they are the source of bis - retinoids and lipofuscin in the retina. bis -Retinoids  constitute the major components of lipofuscin, a complex fluorescent mixture of cellular debris that accumulates with age in the lysosomal compartment of the RPE. bi s-Retinoids, as well as lipofuscin, display broad toxicity, including photoreactivity and inhibition of lysosomal function among others.



The perception of light is possible because of molecules like rhodopsin in which a protein known as opsin which is a g-protein coupled receptor binds to a chromophore molecule known as retinol which attaches to a lysine amino acid in the seventh transmembrane region. Retinol possesses a specific conformation because of a double bond which exists insists between the 11th and 12th carbons in the chain when light is absorbed of a specific wavelength the electrons can move allowing this double bond to rotate and now the retinol molecule exists in trans while the cysts form of retinol binds to opsin to form rhodopsin the transform will dissociate from opsin and will undergo a series of enzymatic reactions before the cysts form is returned and it can bind again to opsin this period in which the chromophore dissociates from opsin and needs to regenerate is known as photo bleaching and for rhodopsin requires about 45 minutes to fully recover in mammals



When retinol changes its shape and dissociates from the binding site of opsin undergoes a conformational shaped change to become meta rhodopsin which activates the g-protein associated with it transducin when the g-protein transducin is activated it dissociates and it's alpha subunit then activates the enzyme phosphodiesterase breaks down cyclic GMP there are sodium channels which require cyclic GMP to stay open once phosphodiester breaks down cyclic GMP these sodium channels closed when the sodium gates closed less positive sodium comes into the cell and the cell becomes more negative it hyperpolarizes as the neuron hyperpolarizes the change in its transmembrane potential then closes voltage regulated calcium gates the decrease in calcium influx then decreases the exocytosis of vesicles of neurotransmitter this change in the release of neurotransmitter then affects the postsynaptic cells depolarizing some and hyperpolarizing other depending on the cell type




In the retina of the eye our photoreceptor cells called rod cells in the outer segment of the rods are stacks of membranous discs and embedded in the disk membrane is the photoreceptor protein rhodopsin bound within rhodopsin is the light-sensitive molecule retina in the outer membrane of the rod RC GMP gated sodium channels while cGMP is bound they remain open and sodium can enter the rod depolarizing  the cell membrane when a photon of light strikes rhodopsin retinal isomerization initiates read ops and activation active rhodopsin binds transducin a trimeric g protein g alpha moves away to another membrane bound protein cGMP phosphodiesterase or PDE when g alpha binds PDE converts the available supply of cGMP to its non cyclic form v prime GMP as cGMP dissociates from sodium channels and is converted to v prime GMP the sodium channels closed when sodium can no longer flow into the cell the membrane hyperpolarizes sending a signal down the cell to its synaptic terminal where the signal begins its journey to the visual centers in the brain

Parts required in phototransduction:

Rhodopsin  Rhodopsin is an essential G-protein coupled receptor in phototransduction.
Retinal Schiff base cofactor All-trans-retinal is also an essential component of type I, or microbial, opsins such as bacteriorhodopsinchannelrhodopsin, and halorhodopsin.
Guanosine diphosphate ( GDP ) 
Guanosine triphosphate GTP
Cyclic guanosine monophosphate (cGMP) 
cGMP-gated channel of rod photoreceptors
Transducin
phosphodiesterase (PDE)
Cyclic nucleotide-gated Na+ ion channels

Parts required to restore the initial state of rhodopsin: 

Guanylate cyclase
Rhodopsin kinase
Arrestin



The steps, or signal transduction pathway, in the vertebrate eye's rod and cone photoreceptors are then: 3

1.The rhodopsin or iodopsin in the disc membrane of the outer segment absorbs a photon, changing the configuration of a retinal Schiff base cofactor inside the protein from the cis-form to the trans-form, causing the retinal to change shape.

2.This results in a series of unstable intermediates, the last of which binds stronger to the G protein in the membrane and activates transducin, a protein inside the cell. This is the first amplification step – each photoactivated rhodopsin triggers activation of about 100 transducins. (The shape change in the opsin activates a G protein called transducin.)

3.Each transducin then activates the enzyme cGMP-specific phosphodiesterase (PDE).

4.PDE then catalyzes the hydrolysis of cGMP to 5' GMP. This is the second amplification step, where a single PDE hydrolyses about 1000 cGMP molecules.

5.The net concentration of intracellular cGMP is reduced (due to its conversion to 5' GMP via PDE), resulting in the closure of cyclic nucleotide-gated Na+ ion channels located in the photoreceptor outer segment membrane.

6.As a result, sodium ions can no longer enter the cell, and the photoreceptor outer segment membrane becomes hyperpolarized, due to the charge inside the membrane becoming more negative.

7.This change in the cell's membrane potential causes voltage-gated calcium channels to close. This leads to a decrease in the influx of calcium ions into the cell and thus the intracellular calcium ion concentration falls.

8.A decrease in the intracellular calcium concentration means that less glutamate is released via calcium-induced Exocytosis to the bipolar cell. (The decreased calcium level slows the release of the neurotransmitter glutamate, which can either excite or inhibit the postsynaptic bipolar cells.)

9.Reduction in the release of glutamate means one population of bipolar cells will be depolarized and a separate population of bipolar cells will be hyperpolarized, depending on the nature of receptors (ionotropic or metabotropic) in the postsynaptic terminal.

Professor Behe described the process whereby the human eye sees: 1

1. Let us return to the question, how do we see? Although to Darwin the primary event of vision was a black box, through the efforts of many biochemists an answer to the question of sight is at hand. When light strikes the retina a photon is absorbed by an organic molecule called 11-cis-retinal, causing it to rearrange within picoseconds to trans-retinal.


2. The change in shape of retinal forces a corresponding change in shape of the protein, rhodopsin, to which it is tightly bound , to metarhodopsin II. As a consequence of the protein’s metamorphosis, the behavior of the protein changes in a very specific way. The altered protein can now interact with another protein called transducin. Before associating with rhodopsin, transducin is tightly bound to a small organic molecule called Guanosine diphosphate ( GDP )  , but when it binds to rhodopsin the GDP dissociates itself from transducin and a molecule called Guanosine triphosphate GTP, which is closely related to, but critically different from, GDP, binds to transducin. The exchange of GTP for GDP in the transducin/rhodopsin complex alters its behavior. 

3. GTP-transducin/rhodopsin binds to a enzyme called Phosphodiesterase ( PDE ) also called  cyclic nucleotide (cNMP) , located in the inner membrane of the cell. 

4. When bound by rhodopsin and its entourage, the phosphodiesterase acquires the ability to chemically cleave a molecule called  Cyclic guanosine monophosphate (cGMP) . Initially there are a lot of cGMP molecules in the cell, but the action of the phosphodiesterase lowers the concentration of cGMP. Activating the phosphodiesterase can be likened to pulling the plug in a bathtub, lowering the level of water.

5. A second membrane protein which binds cGMP, called an ion channel, can be thought of as a special gateway regulating the number of sodium ions in the cell. The ion channel normally allows sodium ions to flow into the cell, while a separate protein actively pumps them out again. The dual action of the ion channel and pump proteins keeps the level of sodium ions in the cell within a narrow range. 


6. When the concentration of cGMP is reduced from its normal value through cleavage by the phosphodiesterase, many channels close, resulting in a reduced cellular concentration of positively charged sodium ions. 

7. This causes an imbalance of charges across the cell membrane which, finally, causes a current to be transmitted down the optic nerve to the brain: the result, when interpreted by the brain, is vision.

Restoring of the initial state : 

If the biochemistry of vision were limited to the reactions listed above, the cell would quickly deplete its supply of 11-cis-retinal and cGMP while also becoming depleted of sodium ions. Thus a system is required to limit the signal that is generated and restore the cell to its original state; there are several mechanisms which do this. Normally, in the dark, the ion channel, in addition to sodium ions, also allows calcium ions to enter the cell; calcium is pumped back out by a different protein in order to maintain a constant intracellular calcium concentration. However, when cGMP levels fall, shutting down the ion channel and decreasing the sodium ion concentration, calcium ion concentration is also decreased. The phosphodiesterase enzyme, which destroys cGMP, is greatly slowed down at lower calcium concentration. Additionally, a protein called guanylate cyclase begins to resynthesize cGMP when calcium levels start to fall. Meanwhile, while all of this is going on, metarhodopsin II is chemically modified by an enzyme called rhodopsin kinase, which places a phosphate group on its substrate. The modified rhodopsin is then bound by a protein dubbed arrestin, which prevents the rhodopsin from further activating transducin. Thus the cell contains mechanisms to limit the amplified signal started by a single photon.

Trans-retinal eventually falls off of the rhodopsin molecule and must be reconverted to 11-cis-retinal and again bound by opsin to regenerate rhodopsin for another visual cycle. To accomplish this trans-retinal is first chemically modified by an enzyme to transretinol, a form containing two more hydrogen atoms. A second enzyme then isomerizes the molecule to 11-cis-retinol. Finally, a third enzyme removes the previously added hydrogen atoms to form 11-cis-retinal, and the cycle is complete.


The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal.
A Ligand Channel through the G Protein Coupled Receptor Opsin (PDF Download Available). Available from: https://www.researchgate.net/publication/23980943_A_Ligand_Channel_through_the_G_Protein_Coupled_Receptor_Opsin [accessed Oct 18 2017]. 10









(A) Schematic of the molecular mechanisms underlying activation. Absorption of a photon (hν) activates a rhodopsin molecule in the disk membrane to R*. Each R* sequentially contacts numerous molecules of transducin (G), catalyzing their activation to G* through exchange of a bound GDP for a GTP from the cytoplasm. Two G*s can bind to each phosphodiesterase holomer, activating the PDE to PDE**. The activated PDE** hydrolyzes cGMP, reducing its cytoplasmic concentration, and thereby causing closure of cGMP-gated channels in the plasma membrane. The rate constant of cGMP hydrolysis by PDE** is indicated by β, and the rate of cGMP synthesis by guanylyl cyclase (GC) is indicated by α. 2


Phototransduction involves three main biochemical events:


Light entering the eye activates the opsin molecules in the photoreceptors


Upon photon absorption, 11-cis-retinal undergoes an isomerization to the all-trans form, causing a conformational change in the rhodopsin. The activated rhodopsin is called metarhodopsin II.

The precursor for 11-cis-retinal is all-trans-retinol (vitamin A). A diet rich in vitamin A is crucial for vision, since vitamin A cannot be synthesized by humans.

Activated rhodopsin causes a reduction in the cGMP intracellular concentration


The cytoplasmic cGMP levels are controlled by cGMP phosphodiesterase, an enzyme that breaks down cGMP. In the dark, the activity of this enzyme is relatively weak. When the photoreceptor is exposed to light, metarhodopsin II stimulates the activity of cGMP phosphodiesterase via transducin, a G protein. GDP-bound inactive transducin will exchange GDP for GTP following interaction with activated rhodopsin. GTP-bound active transducin will increase the activity of cGMP phosphodiesterase. The result is decreased levels of cGMP in the cytoplasm.



Chemistry and Biology of the Initial Steps in Vision 5

Visual transduction is the process in the eye whereby absorption of light in the retina is translated into electrical signals that ultimately reach the brain. The first challenge presented by visual transduction is to understand its molecular basis. We know that maintenance of vision is a continuous process requiring the activation and subsequent restoration of a vitamin A–derived chromophore through a series of chemical reactions catalyzed by enzymes in the retina and retinal pigment epithelium (RPE). Diverse biochemical approaches that identified key proteins and reactions were essential to achieve a mechanistic understanding of these visual processes. The three-dimensional arrangements of these enzymes' polypeptide chains provide invaluable insights into their mechanisms of action. A wealth of information has already been obtained by solving high-resolution crystal structures of both rhodopsin and the retinoid isomerase from pigment RPE (RPE65). Rhodopsin, which is activated by photoisomerization of its 11-cis-retinylidene chromophore, is a prototypical member of a large family of membrane-bound proteins called G protein–coupled receptors (GPCRs). RPE65 is a retinoid isomerase critical for regeneration of the chromophore. Electron microscopy (EM) and atomic force microscopy have provided insights into how certain proteins are assembled to form much larger structures such as rod photoreceptor cell outer segment membranes. 

The last 30 years of visual system research have resulted in a greater understanding of cellular signal transduction processes in the eye than in any other organ of human body. Signaling pathways, initiated by activation of rod and cone visual pigments, orchestrate precise changes in cGMP and Ca2+ that act as second messengers to generate electrical signals. In 1994, when at the University of Washington, I asked: “Is vertebrate phototransduction solved? Although the general mechanism of photoactivation is now known in great detail, key steps in the sequence of reactions associated with the quenching, light adaptation, channel function, and restoration of the dark state of photoreceptors remain to be elucidated. Among many questions, a few seem to be critical for understanding phototransduction and related hormonal-transduction systems: How is the rhodopsin structure related to the activated and quiescent states of the receptor? ….” Detailed progress has been made in elucidating G protein inactivation,  rhodopsin kinase and arrestin structure/function, and guanylate cyclase-activating proteins' (GCAPs) regulation of guanylate cycles, among other processes. Although far from complete and quantitative (Fig. 1), today we have a solid conceptual framework for the processes of visual pigment photoactivation, signal amplification, and quenching of the phototransduction cascade.

   

Phototransduction in a rod outer segment. Phototransduction can be described in three stages shown from top to bottom in this cartoon. 
When light strikes rhodopsin (red), it causes isomerization of the 11-cis-retinal chromophore to an all-trans configuration and a conformational change in the opsin protein. This, in turn, leads to formation of a complex with the heterotrimeric G protein, transducin. Nucleotide exchange in the transducin α-subunit from guanosine diphosphate to GTP causes dissociation of transducin with formation of the transducin α-subunit. This subunit interacts with tetrameric cGMP–specific PDE, whereas the transducin βγ-subunit complexes with phosducin. One activated rhodopsin molecule can activate dozens of transducin molecules in this first amplification stage of phototransduction. Displacement of the inhibitory γ-subunit activates PDE in the second amplification step of phototransduction. The resulting decrease in the concentration of cGMP is associated with a decrease in intradiscal Ca2+ concentration because cGMP is a ligand for cGMP-gated cation channels (shown in blue in the plasma membrane), nonselective channels that also allow passage of Ca2+ in their cGMP-bound state. The low Ca2+-level is maintained by the light-insensitive Na+/Ca2+-K+ exchanger, which extrudes Ca2+ ions out against a gradient in exchange for Na+ and K+ ions. Each of the above-activated molecules needs to return to its inactive state before absorption of the next photon. Thus, rhodopsin is phosphorylated at its C-terminus by GRK1 (or rhodopsin kinase [RK]), followed by binding of arrestin, a capping protein. Guanosine triphosphate is hydrolyzed by the α-subunit of transducin with the help of a GTPase-activating protein. Guanylate cyclase 1 and GC2 (GC, light/dark-brown box) are activated by Ca2+-binding proteins (GCAP1 and GCAP2, black ball) in their Ca2+-free forms to restore cGMP levels and open the cyclic nucleotide–gated cation channels in the plasma membrane. Guanylate cyclase-activating proteins are inactivated and GC activities return to their dark condition. Once GTP is hydrolyzed by the α-subunit of transducin along with phosphorylation of phosducin, the heterotrimeric G protein is restored. Opsin recombines with 11-cis-retinal and the rhodopsin thus formed is ready to be photoactivated. 
Note that all these processes take place on the cytoplasmic surfaces of disc and plasma membranes.

Amplification in the phototransduction cascade  4


The activation of a single rhodopsin by a single photon is sufficient to cause a significant change in the membrane conductance. This is possible due to amplification steps present in the transduction cascade.

A single photoactivated rhodopsin catalyses the activation of 500 transducin molecules. Each transducing can stimulate one cGMP phosphodiesterase molecule and each cGMP phosphodiesterase molecule can break down 1000 molecules of cGMP per second. Therefore, a single activated rhodopsin can cause the hydrolysis of more than 100.000 molecules of cGMP per second. 

Today, as a result of the work of Evgeniy Fesenko and co-workers at the Academy of Sciences in Moscow, Denis Baylor at Stanford University, KingWai Yau of the University of Texas, and others, we are much closer to understanding the linkage between the bleaching and the closing of the sodium pores. For example, it had been very hard to imagine how the bleaching of a single molecule could lead to the closing of the millions of pores that the observed potential changes would require. It now appears that the pores of the receptor are kept open by molecules of a chemical called cyclic guanosine monophosphate, or cGMP. When the visual pigment molecule is bleached a cascade of events is let loose. The protein part of the bleached pigment molecule activates a large number of molecules of an enzyme called transducin; each of these in turn inactivates hundreds of cGMP molecules, with consequent closing of the pores. Thus as a result of a single pigment molecule being bleached, millions of pores close off. 6

The light-sensitive properties of rhodopsin, the vertebrate rod photoreceptor pigment, originate from its 11- cis -retinal chromophore. The absorption of light by rhodopsin results in the isomerization of the 11- cis -retinal chromophore to all- trans forming the enzymatically active intermediate, metarhodopsin II, which commences the visual transduction process. Photoisomerization of the 11- cis -retinal chromophore to all- trans renders rhodopsin insensitive to light. To regain sensitivity to light, all- trans -retinal has to be removed and fresh 11- cis -retinal be supplied to regenerate rhodopsin. These reactions take place in the outer segment of the rod photoreceptor, where rhodopsin is densely packed. Removal of all- trans -retinal begins with the hydrolysis of the Schiff base that links it covalently to opsin, the protein part of rhodopsin. All- trans -retinal is then released, leaving behind opsin, which can combine with fresh 11- cis -retinal to regenerate rhodopsin. The removal of all- trans -retinal is completed through reduction to all-trans - retinol in a reaction catalyzed by the enzyme retinol dehydrogenase  and using NADPH as a cofactor. Subsequently, in a process facilitated by the interphotoreceptor retinoid-binding protein (IRBP), all- trans -retinol leaves the rod outer segment and is transferred to the adjacent cells of the retinal pigment epithelium (RPE). There, all- trans -retinol enters a series of reactions that culminate in the generation of 11- cis -retinal. Another source of all- trans -retinol is the choroidal circulation, which delivers it bound on retinol-binding protein (RBP) to the basal side of the RPE cells. Inside the RPE cells, all-trans - retinol is esterified by lecithin/ retinol acyltransferase (LRAT) to form retinyl esters, which are the substrate used by the RPE65 protein to make 11- cis -retinol. Retinol dehydrogenase RDH5 oxidizes 11- cis -retinol to 11- cis -retinal, which is then transported from the RPE cells to rod outer segments by IRBP.

Because of its importance for maintaining light sensitivity, rhodopsin regeneration has been and is being studied extensively at the molecular, cellular, and whole organism levels. Additional importance of the reactions involved in rhodopsin regeneration stems from the involvement of the highly reactive allylic aldehydes 11- cis and all- trans -retinal. The cytotoxic properties of all-trans -retinal have been extensively documented and studied; on the basis of its structure, the toxic effects of 11- cis -retinal would be expected to be similar. The cytotoxic potential of 11- cis and all-trans - retinal extends even further, as they are the source of bis - retinoids and lipofuscin in the retina. bis -Retinoids  constitute the major components of lipofuscin, a complex fluorescent mixture of cellular debris that accumulates with age in the lysosomal compartment of the RPE. bi s-Retinoids, as well as lipofuscin, display broad toxicity, including photoreactivity and inhibition of lysosomal function among others.

Retinal is attached by a Schiff base linkage to the ε-amino group of a Lysine side chain in the middle of the 7th helix, and this retinal Schiff base (RSB) is protonated (RSBH + ) in most cases.


Figure 1. Signal amplification in rod phototransduction. 11
(A) Three distinct biochemical stages amplify the signal generated by a single activated rhodopsin molecule, R*: (1) high rate of transducin activation (Gαβγ); (2) high rate of cGMP hydrolysis by each activated PDE molecule; and (3) cooperative gating of the cGMP-sensitive ion channels by cGMP. 
(B) The time course of the electrical response to a single photon (thick gray trace) is compared with the time course and number of the active transducin-PDE complexes (thin black trace). 
(C) The spatial profile of the change in cGMP concentration relative to its dark level, at 3 times indicated at the time points in panel B. A schematic representation of the rod cell is shown beneath the graph. Note that the number of active transducin-PDE complexes at any time is quite small (Panel B) and the relative change in cGMP is likewise rather modest. Panel A is adapted with permission from ref. 2; panel B is adapted with permission from ref. 14.





Among the fastest of all responses mediated by a G Protein Coupled Receptor ( rhodopsin) , is the response of the eye to light: it takes only 20 msec (  A millisecond is a thousandth of a second.) for the most quickly responding photoreceptor cells of the retina (the cone photoreceptors, which are responsible for color vision in bright light) to produce their electrical response to a sudden flash of light.

This exceptional speed is achieved in spite of the necessity to relay the signal over multiple steps of an intracellular signaling cascade.

A single photoactivated rhodopsin catalyses the activation of 500 transducin molecules. Each transducing can stimulate one cGMP phosphodiesterase molecule and each cGMP phosphodiesterase molecule can break down 1000 molecules of cGMP per second. Therefore, a single activated rhodopsin can cause the hydrolysis of more than 100.000 molecules of cGMP per second.

But photoreceptors also provide a beautiful illustration of the positive advantages of intracellular signaling cascades: iparticular, such cascades allow spectacular amplification of the incoming signal and also allow cells to adapt so as to be able to detect signals of widely varying intensity.

In this photoreceptor cell, light is sensed by rhodopsin, a G-protein-coupled light receptor. Light-activated rhodopsin activates a G protein called transducin. The activated α subunit of transducin then activates an intracellular signaling cascade that causes cation channels to close in the plasma membrane of the photoreceptor cell. This produces a change in the voltage across the cell membrane, which alters neurotransmitter release and ultimately leads to a nerve impulse being sent to the brain.

When lighting conditions are dim, as on a moonless night, the amplification is enormous: as few as a dozen photons absorbed in the entire retina will cause a perceptible signal to be delivered to the brain. In bright sunlight, when photons flood through each
photoreceptor cell at a rate of billions per second, the signaling cascade undergoes a form of adaptation, stepping down the amplification more than 10,000-fold, so that the photoreceptor cells are not overwhelmed and can still register increases and decreases in the strong light. The adaptation depends on negative feedback: an intense response in the photoreceptor cell decreases the cytosolic Ca2+ concentration, inhibiting the enzymes responsible for signal amplification.

Adaptation frequently occurs in intracellular signaling pathways that respond to extracellular signal molecules, allowing cells to respond to fluctuations in the concentration of such molecules regardless of whether
they are present in small or large amounts. By taking advantage of positive and negative feedback mechanisms (see Figure 16–14), adaptation thus allows a cell to respond both to messages that are whispered and to
those that are shouted.

The signal from activated visual pigment (rhodopsin in rods or cone opsins in cones) must be sufficiently amplified to generate an electrical response that overcomes intrinsic noise. 12  Transduction from the light-absorbing visual pigment into an electrical response utilizes a G protein signaling pathway termed the phototransduction cascade, which leads to a decrease in the second messenger cGMP and the closure of cGMP-sensitive cation channels. The resulting hyperpolarization transiently decreases the release of glutamate from the photoreceptor synaptic terminals, signaling the number of absorbed photons to the rest of the visual system. Remarkably, photoreceptors both detect low light levels (single photons in the case of rods) and continue to rapidly and reliably signal changes in light intensity as illuminance increases over 10 orders of magnitude during the course of a typical day.

The photoresponse persists until each phototransduction protein becomes deactivated through the action of one or more regulatory enzymes One necessary reaction is catalyzed by a triumvirate complex of proteins consisting of RGS9-1, Gβ5-L, and R9AP

Question: Had these protein complexes which by their own have no function, not have emerge together and right in the beginning, in order to provide these highly complex regulation functions?


Cornelius Hunter:
New research has discovered ciliary photoreceptor cells providing directional light detection in the brachiopod Terebratalia transversa. This unexpected finding further complicates the already circuitous evolutionary narrative of the origins of vision at the cellular level.

At the molecular level vision begins with a complex signal transduction cascade. As photons enter your eye they interact with light-sensitive chromophore molecules in the photoreceptor cells. The interaction causes the chromophore to change configuration and this, in turn, influences the large, trans-membrane rhodopsin protein to which the chromophore is attached.

The photocell’s cellular signal transduction cascade which is initiated by a photon interacting with a light-sensitive chromophore molecule known as retinal. The interaction alters the electron distribution of the retinal molecule, thus making intricate changes to its force field which influences several amino acids of the large, trans-membrane opsin protein to which the chromophore is attached. 8

The Vision Cascade is Initiated Not by Isomerization but by Force Field Dynamics  9
As you read these words a frenzy of activity is taking place as the light entering your eye triggers a highly detailed sequence of actions, ultimately causing a signal to be sent to your brain. In fact, even a mere single photon can be detected in your vision system. It all starts with a photon interacting with a light-sensitive chromophore molecule known as retinal. The interaction alters the retinal molecule and this, in turn, influences the large, trans-membrane opsin protein to which the chromophore is attached. This is just the beginning of the cellular signal transduction cascade. In the next step the opsin causes the activation of hundreds of transducin molecules. These, in turn, cause the activation of cGMP phosphodiesterase (by removing its inhibitory subunit), an enzyme that degrades the cyclic nucleotide, cGMP.

A single photon can result in the activation of hundreds of transducins, leading to the degradation of hundreds of thousands of cGMP molecules. cGMP molecules serve to open non selective ion channels in the membrane, so reduction in cGMP concentration serves to close these channels. This means that millions of sodium ions per second are shut out of the cell, causing a voltage change across the membrane. This hyperpolarization of the cell membrane causes a reduction in the release of neurotransmitter, the chemical that interacts with the nearby nerve cell, in the synaptic region of the cell. This reduction in neurotransmitter release ultimately causes an action potential to arise in the nerve cell.

New research is now helping to explain the details of the first step in this Rube Goldberg machine. What happens when the photon interacts with the retinal molecule? And how does this influence the opsin protein? It had been thought that the key step was a change in the structural configuration of the chromophore. This photoisomerization is caused by the photon and was thought to be how the chromophore influences the opsin.

The new research, however, found that when isomerization is disabled the vision cascade continues to function normally. It seems that a key step, occurring before isomerization, is a shift in the electron distribution of the chromophore. This shift modifies the electric field surrounding the molecule, and this in turn influences several amino acids of the opsin protein, which in turn leads to the activation of the transducin molecules. 

Does evolution shape force fields?

The new research presents a different problem for evolution. In addition to designing the opsin protein, evolution must now design the electric field surrounding the chromophore, and how it responds to photon interaction. And while it is busy with that task, it must also specify the correct amino acids at the correct locations within the opsin, that will be influenced by the chromophore’s dynamic electric field.

This massive design problem involves what is known as an n-body solution. That is, the various sub atomic particles in the opsin amino acids and the chromophore, including the chromophore’s flowing electrons which respond to the photon, all contribute to the environmental force fields.

Modeling these force fields and how molecules respond to them is a major problem in molecular dynamics studies. Both the modeling of the force fields, and the molecular dynamics is challenging and computationally intensive. For instance, each particle influences each of the other particles. And as a particle moves, all of its influences change. But other particles are moving as well, so the dynamics quickly become extremely complicated.

The previous model, which had evolution designing the chromophore and its photoisomerization, was complicated enough. Now evolution must also design force fields and their dynamics caused by electron flow within the chromophore. The design space just took another quantum leap.


The chromophore photoisomerization is the beginning of a remarkable cascade that causes action potentials to be triggered in the optic nerve. In response to the chromophore photoisomerization, the rhodopsin causes the activation of hundreds of transducin molecules. These, in turn, cause the activation of cGMP phosphodiesterase (by removing its inhibitory subunit), an enzyme that degrades the cyclic nucleotide, cGMP.
A single photon can result in the activation of hundreds of transducins, leading to the degradation of hundreds of thousands of cGMP molecules. cGMP molecules serve to open non selective ion channels in the membrane, so reduction in cGMP concentration serves to close these channels. This means that millions of sodium ions per second are shut out of the cell, causing a voltage change across the membrane. This hyperpolarization of the cell membrane causes a reduction in the release of neurotransmitter, the chemical that interacts with the nearby nerve cell, in the synaptic region of the cell. This reduction in neurotransmitter release ultimately causes an action potential to arise in the nerve cell.

This is the beginning of vision. And while there are variations on this remarkable sequence, it is found throughout the wide variety of vision systems found in biology. 7

Over and over, the fascinating designs found in biology must have, according to evolution, appeared early on, even before any need for such marvels.
For vision, this theme of early complexity is repeated at the cellular level, where two distinct photoreceptor cell morphologies—rhabdomeric and ciliary—are found. These two morphologies have different membrane folding strategies as well as biochemical pathways. But their widespread presence in organisms forces evolutionists to conclude they both must have been present in the last common bilaterian ancestor. Rhabdomeric photoreceptor cells are often associated with invertebrates and ciliary photoreceptor cells with vertebrates, but both invertebrates and vertebrates have cells with both morphologies.

And so both morphologies must trace back to that last common bilaterian ancestor. While it may stretch common sense for early evolution to create such complexity in duplicate, if you believe it can perform the feat once, then why not twice?

But there is more to the story. In invertebrates the ciliary morphology plays a lesser role. It is not found to provide directional light detection, but in more rudimentary light detection roles. So how then does it emerge as the chief architecture in vertebrate vision systems? The evolutionary narrative calls for a migration of the ciliary photoreceptor cells to the retina where they overtake the rhabdomeric photoreceptor cells while attaining new visionary skills. Why (and how) this would happen is anyone’s guess.
Only a few years ago proponents of evolution were confident of this narrative. It was, according darwinists, a compelling story that reaffirmed the truth of evolution. These amazing claims were yet another demonstration of how darwinists interpret unlikely data into a favorable apologetic.
But now the story has become even more unlikely. The new research has indeed found ciliary photoreceptor cells providing directional light detection in Terebratalia transversa, an invertebrate. The narrative of ciliary photoreceptor cells migrating to the retina in vertebrates suddenly makes little sense. Evolution needs a new narrative, and as usual it is more complex:
The presence of ciliary photoreceptor-based eyes in protostomes suggests that the transition between non-visual and visual functions of photoreceptors has been more evolutionarily labile than previously recognized, and that co-option of ciliary and rhabdomeric photoreceptor cell types for directional light detection has occurred multiple times during animal evolution.
In other words, yes that evolutionary scenario we were so confident of must be discarded, but so what? We can always add more drama to the plot line. Whatever biology reveals, it must have evolved—theory respectability is not important. Religion drives science, and it matters.

Cornelius Hunter:
The incredible cellular signal transduction cascade at the heart of vision evolved from a similar system in yeast. Yes, believe it or not, it is that simple. As they explain:

In yeast (a fungus), these receptors are sensitive to pheromones, and they even direct a signal through proteins homologous to non-opsin phototransduction proteins. As such, a signaling pathway exists outside animals, which is very similar to phototransduction, except that the receptor protein detects pheromones, not light. Receptors outside animals share some characteristics with opsin, like snaking through a cell membrane seven times. It is one of these serpentine proteins that served as the progenitor of the first opsin protein, as evidenced by the similarity of opsins and other serpentine proteins.

There you have it. The eye evolved from a signaling pathway outside of animals. The evolutionists triumphantly conclude that:

the gap in understanding of the molecular evolution of eye components is all but closed, highlighting the bankruptcy of the argument that design is required to explain the origins of biological features

The natural processes of mutation (especially mutations that duplicate genes) and selection have left clear traces as causal agents of the evolution of eyes.  3



1. https://uncommondescent.com/intelligent-design/could-the-eye-have-evolved-by-natural-selection-in-a-geological-blink/
2. http://www.sciencedirect.com/science/article/pii/S0896627300000635
3. https://en.wikipedia.org/wiki/Photoreceptor_cell
4. https://openwetware.org/wiki/BIO254:Phototransduction
5. http://iovs.arvojournals.org/article.aspx?articleid=2166602
6. http://hubel.med.harvard.edu/book/b11.htm
7. http://darwins-god.blogspot.com.br/2011/03/unexpected-role-for-ciliary.html
8. http://darwins-god.blogspot.com.br/2011/12/new-book-doesnt-explain-how-eyes.html
9. http://darwins-god.blogspot.com.br/2011/06/vision-cascade-is-initiated-not-by.html
10. https://www.researchgate.net/publication/23980943_A_Ligand_Channel_through_the_G_Protein_Coupled_Receptor_Opsin
11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160332/
12. http://www.jbc.org/content/287/3/1620.full

http://darwins-god.blogspot.com.br/2010/02/early-vision-more-complicated.html



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4 Rhodopsin , opsins , and retinol on Fri Oct 13, 2017 7:41 pm

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Rhodopsin , opsins , and retinol


Rods and cones are photoreceptive cells located in the retina of the eye. The outer segment contains the phototransduction apparatus, shown here for a rod.

Opsin—the protein that underlies all animal vision., has become a favorite research target, not only of vision scientists but of many researchers interested in the evolution of protein structure, function, and specialization. This level of focus has made the opsins canonical G-protein-coupled receptors (GPCRs) and arguably the most investigated protein group for its evolutionary radiations and diverse functional specializations. 15 Still, opsin’s early evolution remains puzzling, and there are many questions throughout its evolutionary history for which we have partial, but tantalizingly incomplete, answers. Obviously the invertebrates, with their astonishing diversity and with evolutionary hints of the most ancient animals in their genomes, functions, and even body plans, offer the best hope of answering many of these fundamental questions.

Rhodopsin Structure and Activation
Rhodopsin consists of an apoprotein opsin and an inverse agonist ( thats like a mechanism which keeps a switch off ), the 11-cis-retinal chromophore, which is covalently bound through a Schiff base linkage to the side chain of Lys296 of opsin protein. 

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". The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light17
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

Residues important for stabilising the tertiary structure 
(e.g. disulphide bridge (S-S), 
amino-terminal (N) glycosylation sites)
activation/deactivation of photopigments (e.g. carboxyl-terminal (C) phosphorylation sites)
membrane anchorage (e.g. palmitoylation sites)

For visible light absorption, all opsins contain an essential amino acid residue called "counter ion", in addition to a retinal-binding site, Lys296 (in the bovine rhodopsin numbering system), where chromophore retinal covalently binds through a protonated Schiff base linkage . The proton on the Schiff base is necessary for visible light absorption, but energetically unstable within the opsin molecule. In opsin pigments, a negatively charged amino acid residue, counterion, stabilizes the protonated Schiff base, and is an essential amino acid residue for opsin pigments to absorb visible light.

Various types of opsin-based pigments with absorption maxima in the visible light region possess a “protonated” Schiff base linkage. In the protein moiety, the positive charge on the protonated Schiff base is unstable, and therefore a counterion, a negatively charged amino acid residue is needed to stabilize the positive charge. In vertebrate visual pigment, glutamic acid at position 113 serves as the counterion 11

Furthermore: movement of the cytoplasmic end of the sixth transmembrane helix is essential for pigment activation. 

In the dark, this Schiff base is protonated, which results in a 498 nm absorption peak. Light activation, however, stimulates chromophore isomerization from 11-cis-retinal to all-trans-retinal and Schiff base deprotonation, accompanied by a spectral shift towards shorter wavelengths, changing the λmax from 498 nm to 380 nm (Fig. 4.1a). Retinal isomerization also initiates structural changes in the rhodopsin polypeptide chain leading through several intermediate states including Batho, Lumi and Meta I rhodopsin to an active state conformation called Meta II. These changes include rearrangements in several structurally important microdomains that are conserved in class A GPCRs such as the D[ERY] motif located in TM3 and the NPxxY motif in helix TM7. Changes in the D[E]RY motif lead to the disruption of an “ionic lock” between Arg135 on TM3 and Glu247 on TM6 and to rearrangements in the NPxxY motif. These atomic alterations result in the movement of transmembrane helix TM5 towards TM6 and the outward tilt of TM6 that opens up the cytoplasmic surface and provides a binding site for its prototypical G protein, transducin, allowing signal transduction to occur. light stimulated transition from dark state rhodopsin to its activated state causes a structural relaxation upon retinal isomerization, which then tightens upon transducin binding. The atomic structure of rhodopsin coupled with radiolytic footprinting led to the identification of ordered waters within transmembrane helices located close to highly conserved and functionally important receptor residues. Photo- stimulated structural rearrangements of the protein backbone and side chains are accompanied by reorganization of these structural water molecules that provide a hydrogen-bond network linking the ligand binding site to the effector (G protein, GRK, arrestin) binding site, indicating that these waters are essential not only for structural stabilization of the receptor but also for the activation process

The placozoan opsins cannot bind retinal, because they lack the amino acid to which retinal binds (amino acids are the building blocks of proteins). Without 'lysine-296', it is unlikely that the placozoan opsins can detect light.


Fig. 4.1 Schematic organization of the retinal rod photoreceptor cell.
(a) Different photo-states of rhodopsin.
(b) Rod photoreceptor cell. Important cell segments and cellular components are indicated.
(c) Schematic organization of rhodopsin dimers in a single rod outer segment disc

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin* 10
Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.

Rhodopsin has the 11-cis-retinal chromophore covalently bound in its protein moiety acting as an inverse agonist in the dark, and light absorption causes the generation of the agonist all-trans-retinal through cis-trans photoisomerization of the chromophore. The covalent bond is formed between the aldehyde group of the retinal and the ϵ-amino group of lysine 296 located at the transmembrane helix VII of rhodopsin, through a protonated Schiff base linkage.

Formation of the protonated Schiff base is an important structural feature for photoreception, because the protonation of the Schiff base causes electron delocalization of the chromophore, resulting in a red shift of the absorption spectrum of rhodopsin, which allows it to absorb visible light

Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins 8

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. To test for the existence of such a constraint, we asked whether it were possible to relocate the highly conserved Lys296 in the visual pigment bovine rhodopsin. 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.

The retinylidene proteins are integral membrane proteins that covalently bind a retinal chromophore. Amino acid sequence comparison divides these proteins into two families known as type I and type II rhodopsins (1). Type I rhodopsins, such as bacteriorhodopsin from the archaeon Halobacterium salinarum, function as light-driven ion transporters, channels, and phototaxis receptors. Type II rhodopsins, best known for the visual pigment of mammalian rod photoreceptor cells, function primarily as photosensitive receptor proteins in metazoan eyes and in certain extraocular tissues. Henceforth, we will use the term “rhodopsin” to refer to the visual pigment of bovine rod photoreceptor cells and “bacteriorhodopsin” to refer to the light-driven proton pump of H. salinarum.

Rhodopsin is a prototypical member of the large family of G protein-coupled receptors (GPCRs; specifically class A GPCRs). It is composed of an apoprotein (called “opsin”) and an 11-cis-retinal chromophore. The GPCR fold comprises seven transmembrane α-helices oriented in a particular spatial arrangement with a specific connectivity

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.

We have tested the hypothesis that the function of rhodopsin (in terms of binding retinal, formation of a long-wavelength pigment, and activation of transducin) constrains the active-site Lys to a location in TM7. The mutants clearly demonstrate that rhodopsin can retain function when the Lys is moved to a different location.

The Evolution of Opsins
T H Oakley and D C Plachetzki,

Opsin genes were very often duplicated and retained during animal evolution. Early opsin gene duplications led to the major opsin groups and more recent duplications mostly led to additional specializations, such as the ability for color vision. As members of highly coordinated protein networks, changes in opsin proteins are sometimes correlated with changes in partnering proteins. The interaction of two evolutionary processes has resulted in the diversity of opsin-based phototransduction pathways observed today that contains a combination of shared and distinct interactions. First, co-option refers to instances where an opsin recruited different intracellular signaling components than its ancestor during evolution. Second, coduplication involved the simultaneous duplication of multiple genes of an ancestral network. Co-option and coduplication are not discrete alternatives; instead, some genes of a network originated by co-duplication, whereas others joined the network by co-option.

Where is the evidence for these claims ? 

The 11-cis-retinylidene bond is protonated, and Glu113 is the counterion of this linkage. A counterion is essential, as positively charged groups are extremely rare in the TMD of membrane proteins.
 
What Is Rhodopsin?

The word ‘rhodopsin’ originates from the Greek words ‘rhodo’ and ‘opsis’, which indicate rose and sight, respectively. Thus, the classical meaning of rhodopsin is the red-colored pigment in the retinal rods of eyes. The chromophore molecule that absorbs light is retinal, which is the origin of the red color.  The modern meaning of rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Rhodopsins are now found in all domains of life and are classified into two groups

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 sometimes called retinylidene proteins because they bind to a light-activated, non-protein chromophore called retinal (retinaldehyde). Opsins are also in some cases called “rhodopsins”, a name originally given to isolated visual pigments that contained both opsin protein and non-protein chromophore in a time before the two separate components were known. Today, the term “Rhodopsin” is used commonly to describe the opsin expressed in vertebrate rod (dim-light) photoreceptors, and the opsins of certain organismal groups, like bacteria. All opsin proteins are embedded in cell membranes, crossing the membrane seven times. 6

All opsins bind a chromophore: the vertebrate visual and non-visual opsins, the invertebrate Gq-coupled opsins, and the Go-coupled opsins all bind 11-cis-retinal, whereas the photoisomerases and the peropsins bind all-trans-retinal 7


Structures of opsins and of the chromophore retinal. 
(a) A model of the secondary structure of bovine rhodopsin. Amino-acid residues that are highly conserved in the whole opsin family are shown with a gray background. The retinal-binding site (K296) and the counterion position (E113) are marked with bold circles, as is E181, the counterion in opsins other than the vertebrate visual and non-visual ones. C110 and C187 form a disulfide bond. 
(b) The chemical structures of the 11-cis and all-trans forms of retinal. 
(c) The crystal structure of bovine rhodopsin (Protein DataBank ID: 1U19 [PDBU19]). The chromophore 11-cis-retinal, K296 and E113 are shown in stick representation in the ringed area. (d) The structure of the Schiff base linkage formed by retinal within the bovine opsin, together with the counterion that stabilizes it.




Residues important for stabilising the tertiary structure
- (e.g. disulphide bridge (S-S),
- amino-terminal (N) glycosylation sites)
- activation/deactivation of photopigments (e.g. carboxyl-terminal (C) phosphorylation sites)
- membrane anchorage (e.g. palmitoylation sites)

These opsins share less than 20 per cent identity between subfamilies.







Fig. 2. Functional diversity of opsin-based pigment. 
Several thousands of opsins are phylogenetically and functionally classified into eight groups. Members of six groups bind 11-cis retinal as a chromophore and form photopigments that activate G protein-mediated signal transductions, and the signaling cascades are classified into two groups, cyclic nucleotide signaling triggered by 


Gt-coupled, 
Gi/o-coupled Go-coupled, 
Gs-coupled  
Go-coupled opsin-based pigments 
phosphoinositol signaling triggered by Gq-coupled opsin-based pigments. 


G protein subtypes that are activated by opsin-based pigments in each group in vitro are also shown on the right side. Members of the remaining two groups, peropsin and retinal photoisomerase groups bind all-trans retinal as a chromophore and light isomerizes it to the 11-cisform. Note that this schematic phylogeny of opsin family is based on the molecular phylogenetic analysis including opsin sequences that were revealed to function as a photopigments and their apparent homologs [8]16



Without rhodopsin and its homologous cone pigments, there would be no image-forming vision.

Water permeation through the internal water pathway in activated GPCR rhodopsin  9

Rhodopsin is a light-driven G-protein-coupled receptor that mediates signal transduction in eyes. Internal water molecules mediate activation of the receptor in a rhodopsin cascade reaction and contribute to conformational stability of the receptor. However, it remains unclear how internal water molecules exchange between the bulk and protein inside, in particular through a putative solvent pore on the cytoplasmic. Using all-atom molecular dynamics simulations, we identified the solvent pore on cytoplasmic side in both the Meta II state and the Opsin. On the other hand, the solvent pore does not exist in the dark-adapted rhodopsin. We revealed two characteristic narrow regions located within the solvent pore in the Meta II state. The narrow regions distinguish bulk and the internal hydration sites, one of which is adjacent to the conserved structural motif “NPxxY”. Water molecules in the solvent pore diffuse by pushing or sometimes jumping a preceding water molecule due to the geometry of the solvent pore. These findings revealed a total water flux between the bulk and the protein inside in the Meta II state, and suggested that these pathways provide water molecules to the crucial sites of the activated rhodopsin.

Rhodopsin biosynthesis



Molecular assemblies that control rhodopsin transport to the cilia  14

Following  review will focus on the conserved molecular mechanisms for the specific targeting of rhodopsin and rhodopsin-like sensory receptors to the primary cilia. We will discuss the molecular assemblies that control the movement of rhodopsin from the central sorting station of the cell, the trans-Golgi network (TGN), into membrane-enclosed rhodopsin transport carriers (RTCs), and their delivery to the primary cilia and the cilia-derived sensory organelles, the rod outer segments (ROS). Recent studies reveal that these processes are initiated by the synergistic interaction of rhodopsin with the active form of the G-protein Arf4 and the Arf GTPase activating protein (GAP) ASAP1. During rhodopsin progression, ASAP1 serves as an activation platform that brings together the proteins necessary for transport to the cilia, including the Rab11a-Rabin8-Rab8 complex involved in ciliogenesis. These specialized molecular assemblies, through successive action of discrete modules, cooperatively determine how rhodopsin and other rhodopsin-like signaling receptors gain access to primary cilia.

Although their architecture varies, from a simple membrane outgrowth to elaborate specialized organelles, such as the retinal photoreceptor rod outer segments (ROS), their common function is to capture extracellular signals. Together with other components of the signal transduction complexes, sensory receptors are highly concentrated in ciliary membranes allowing for exceptional sensitivity to external stimuli. In the case of vision, the architecture and the molecular composition of the ROS provide for the optimum performance underlying extraordinary light sensitivity, i.e. the light receptor rhodopsin and associated phototransduction machinery concentrated in the stacked disk membranes ensure the capture of a single photon of light

The challenge in maintaining the distinctive light sensitivity of the rod outer segments  ROS is partially met by the tight control of the protein entrance across the base, the connecting cilium, which is equivalent to the transition zone of primary (non-motile) cilia. Rhodopsin constitutes the main ciliary-targeted cargo protein in rod photoreceptors. Its delivery to ROS is an outstanding case of ciliary receptor transport, with connecting cilia trafficking ~1000 rhodopsin molecules per second

Like all membrane proteins, rhodopsin follows the intracellular path from the site of its synthesis, the endoplasmic reticulum (ER) to the Golgi complex responsible for N-linked oligosaccharide modifications, sorting and transport. The ER and the Golgi are localized in the myoid part of the rod inner segment (RIS)(Fig 1A).



Figure 1 (A) Diagram of a photoreceptor cell.
Primary cilium originates from the basal body (BB) in the rod inner segment (RIS) and the axoneme elaborates the rod outer segment (ROS). The transition zone is a “connecting cilium”, the gateway to the ROS. Golgi and the TGN are localized in the myoid region (M) of the RIS. RTCs that bud from the TGN are targeted to the base of the cilium (arrow), through the ellipsoid region (E) filled with mitochondria (m). N, nucleus; Sy, synapse. (B) Molecular interactions taking place during rhodopsin progression to the ciliary base. At the TGN, activated Arf4 interacts with rhodopsin and they recruit ASAP1 into the ternary complex. ASAP1 likely initiates membrane deformation through its BAR domain while mediating GTP-hydrolysis on Arf4, which then dissociates from the TGN. Next, ASAP1 selectively binds Rab11a, which also associates with rhodopsin. ASAP1 and Rab11a recruit Rabin8 and Rab8. On RTCs, ASAP1 serves as a scaffold for the Rab11a/Rabin8/Rab8 complex, which controls the activation of Rab8. Activated Rab8 regulates RTCs fusion and the delivery of rhodopsin across the diffusion barrier surrounding the cilium. Rhodopsin then proceeds through the transition zone (or the connecting cilium) into the ciliary axoneme that forms the ROS.

After exiting the Golgi complex, rhodopsin reaches the trans golgi network (TGN) where it is specifically incorporated into rhodopsin transport carriers (RTCs)

Rhodopsin has two functional ciliary targeting signals, the VxPx and the FR, which are recognized by Arf4 and ASAP1, respectively


One of the main functions of the canonical coat complexes is the recognition of intracellular targeting signals in cargo proteins. Rhodopsin possesses the C-terminal VxPx targeting motif that regulates Arf4 binding and rhodopsin targeting in vivo.


The cytoplasmic C-terminal VxPx motif, which is the Arf4 binding site, is circled.

The intraflagellar transport protein IFT57 is required for cilia maintenance and regulates IFT-particle–kinesin-II dissociation in vertebrate photoreceptors 12

Vertebrate photoreceptors are highly specialized neurons that possess a modified sensory cilium known as the outer segment. The outer segment develops as an extension of a nonmotile primary cilium. As the outer segment lacks the machinery for protein synthesis, all protein destined for the outer segment must pass through the connecting cilium. Large amounts of protein synthesized in the inner segment must be efficiently transported to the outer segment to replenish material lost from the distal tips each day. Estimates from mammalian systems have calculated ∼2000 rhodopsin molecules per minute must be transported to the outer segment to compensate for lost material.  Hence, both the development and survival of the photoreceptor require this continual transport of protein to the outer segment . The C-terminal tail of rhodopsin contains a sorting sequence that is necessary and sufficient for transport to the outer segment.

Mutations in this region result in protein accumulation in the inner segment and at the base of the connecting cilium in mice, rats and frogs leading to photoreceptor degeneration. Indeed, mutations in the C terminus of human rhodopsin, such as P347L and S344Ter, can cause retinitis pigmentosa. It is imperative, therefore, that cargo targeted for the outer segment reach its destination or retinal degeneration will occur. Thus, both mutations within the opsin gene and mutations in the transport machinery can cause retinal degenerative diseases. Protein transport along a ciliary axoneme, such as the connecting cilium, occurs via the process known as intraflagellar transport (IFT)

13


1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858459/
2. http://www.cs.ucsb.edu/~dbl/papers/larusso_jme_2008.pdf
3. http://genomewiki.ucsc.edu/index.php/Opsin_evolution:_orgins_of_opsins
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781858/
5. http://retina.umh.es/webvision/Evolution.%20PART%20I.html#Ciliary photoreceptors in the eyes of extant chordates
6. http://evolutionarynovelty.blogspot.com.br/2008/12/opsins-amazing-evolutionary-convergence.html
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1088937/
8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746867/
9. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176876
10. http://www.jbc.org/content/285/11/8114.full
11. Evolution of Visual and Non-visual Pigments, page 14
12. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2637114/
13. https://sci-hub.bz/https://www.nature.com/articles/nrm952
14. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514645/
15. Evolution of Visual and Non-visual Pigments, page 106
16. http://www.sciencedirect.com/science/article/pii/S000527281300159X#f0010
17. https://sci-hub.bz/http://www.nature.com/nsmb/journal/v11/n3/full/nsmb731.html

Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue
http://europepmc.org/articles/PMC3612648

The common assertion is that eyespots like in Euglena evolved into complex vertebrate eyes. The remarkable thing is, that bacterias and algae use Type 1 opsins, which are not homologous with type 2 opsins, used in vertebrates. So what is the escape? Ahm, yah..... there was CONVERGENCE !!!
So opsins evolved twice independently. Cool, hah ??



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5 Type 1 opsins on Sun Oct 15, 2017 7:38 am

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Type 1 opsins 5

The bilaterian opsin repertoire comprises the c-opsin class and the second ancestral class which has diversified into several subclasses termed as 

rhabdomeric opsins (r-opsins), 
Go-coupled opsins, 
neuropsins 
RGR 

From these, only three groups (namely c-, r- and Go-coupled opsins) seem to have been recruited for visual purposes 5

Presumably the common ancestor of all true opsins lacked the chromophore- binding lysine at position 296 (bovine rod opsin numbering, used throughout the chapter) but nevertheless interacted with a retinoid ligand to initiate signaling. Making the reasonable assumption that the binding was specific to the conformation of this ligand, the most likely form was an all- trans structure (because this is the most common and stable form of retinoids and also the form present in the chromophore 4 a property expected in the ancestral preopsin. Of course, all opsins devoted to light-sensing today have a lysine residue at this position that forms the Schiff base that covalently binds the chromophore. Opsins diversified almost immediately after first appearing, forming three undisputed clades: ciliary (or C-type) opsins, rhabdomeric (or R-type) opsins, and Go-RGR (or Group 4) opsins. 
Indeed, it can be reasonably argued that everything to do with photosensing and vision was invented before the radiation of the major animal groups.



Developing cnidarian eyes even use genetic mechanisms similar to those of higher animals, including vertebrates (Kozmik et al. 2008 ). Based on what we see in the “simple” cnidarians, it would appear that nearly every important function of opsins most likely originated before the first bilaterian animals appeared, and what followed in subsequent animal evolution arose from modifications to the ancestral collection of “urbilaterian” opsins and their associated mechanisms.

So basically, there was no further evolution right after vision appeared !!

Invertebrate opsins bind to a diverse set of G-proteins, and they presumably interact with a correspondingly diverse set of second messenger systems and signaling cascades.

binding pocket when phototransduction is initiated in modern opsins).
Type I opsins 
are present in bacteria and algae, in both prokaryotic and eukaryotic microbes, functioning as light-driven proton pumps, sensory receptors, and in various other unknown functions. They have varied function, including bacterial photosynthesis (bacteriorhodopsin), which is mediated by pumping protons into the cell, and phototaxis (channelrhodopsin), which is mediated by depolarizing the cell membrane.and are referred to by various names, including 

Bacteriorhodopsin 
bacterial sensory rhodopsins
Channelrhodopsin 
halorhodopsin 
proteorhodopsin

Bacteriorhodopsin
Bacteriorhodopsin is a protein used by Archaea, most notably by Halobacteria, a class of the Euryarchaeota. It acts as a proton pump; that is, it captures light energy and uses it to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy.

Discovery of 7 times higher complexity of protein folding!  2
Biophysicists at JILA have measured protein folding in more detail than ever before, revealing behavior that is surprisingly more complex than previously known. . . .
They fold into three-dimensional shapes that determine their function through a series of intermediate states, like origami. Accurately describing the folding process requires identifying all of the intermediate states. The JILA research revealed many previously unknown states by unfolding an individual protein. For example, 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) biophysicist working at JILA, a partnership of NIST and the University of Colorado Boulder. “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.














Channelrhodopsins
Channelrhodopsins are a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light

Channelrhodopsins: light-activated ion channels 1



Channelrhodopsins (ChRs) are light-gated cation channels and function as primary photoreceptors in motile green algae. In the algae, ChRs 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.
 
The first two channelrhodopsins to be identified were the two isoforms from Chlamydomonas reinhardti, namely ChR1 and ChR2. Subsequently, further ChRs were found in related algae including Volvox cateri (VChR1 and VChR2), Dunaliella salina (DChR1) and Mesostigma viride (MChR1). All ChRs share a common architecture comprising an N-terminal membrane-spanning domain and a C-terminal cytosolic domain. Heterologous expression in conjunction with electrophysiological measurements showed that the membrane-spanning domain itself is sufficient to drive photocurrents. Thus, the putative channel is formed by the seven transmembrane helices, whereas the retinal chromophore is bound via a conserved lysine residue in helix seven (Figure 2a).
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).

The Microbial Opsin Family of Optogenetic Tools 3

Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

Diverse and elegant mechanisms enable organisms to harvest light for a variety of survival functions, including energy generation and the identification of suitable environments. A major class of light-sensitive protein consists of 7-transmembrane (TM) rhodopsins that can be found across all kingdoms of life and serve a diverse range of functions



Many motile microorganisms have evolved opsin-based photoreceptors to modulate flagellar beating or flagellar motor rotation and thereby direct phototaxis toward environments with optimal light intensities for photosynthesis. Microbial opsins can be treated as precise and modular photosensitization components for introduction into non-light-sensitive cells to enable rapid optical control of specific cellular processes.

Each opsin protein requires the incorporation of retinal, a vitamin A-related organic photon-absorbing cofactor, to enable light sensitivity; this opsin-retinal complex is referred to as rhodopsin.

The retinal molecule is covalently fixed in the binding pocket within the 7-TM helices and forms a protonated retinal Schiff base (RSBH+; Figure 2A) with a conserved lysine residue located on TM helix seven (TM7). The ionic environment of the RSBH+, heavily influenced by the residues lining the binding pocket, dictates the spectral characteristics of each individual protein; upon absorption of a photon, the retinal chromophore isomerizes and triggers a series of structural changes leading to ion transport, channel opening, or interaction with signaling transducer proteins



Channelrhodopsins (ChRs), another group of microbial rhodopsins, were discovered in green algae where they function as light-gated cation channels within the algal eye to depolarize the plasma membrane upon light absorption (Fig. 1.2 )


Thus, ChRs also naturally function as signaling photoreceptors. The discovery of ChR led to the emergence of a new field, optogenetics (Miesenbock 2011 ), in which light-gated ion channels and light-driven ion pumps are used to
depolarize and hyperpolarize selected cells of neuronal networks. This new method is strongly expected to aid in understand the circuitry of the brain.

1









2











The form and function of channelrhodopsin 9




1. https://www.biologie.hu-berlin.de/de/gruppenseiten/expbp/research/channelrhodopsins-light-activated-ion-channels
2. https://uncommondescent.com/intelligent-design/discovery-of-7-times-higher-complexity-of-protein-folding/
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. http://science.sciencemag.org/content/357/6356/eaan5544.full



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6 The Visual Cycle on Sun Oct 15, 2017 6:05 pm

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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. 22

Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. 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.21

Restoration of light sensitivity requires chemical reisomerization of retinaldehyde via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE). 14 Opsin visual pigments are located in a stacked membranous structure of rod and cone photoreceptor cells called the outer segment (OS).


Schematic representation of phototransduction components in photoreceptors
The outer segment (OS) is a modified cilium, with the basal body (BB) located at the apical region of the inner segment. The transition zone extends from the BB and gives rise to the axoneme, which continues into the outer segment. The disc membranes in the outer segment are enriched in rhodopsin and other proteins required for carrying out the phototransduction cascade. Part of the cascade also takes place in the overlaying RPE (retinal pigmented epithelium). In addition, RPE is also required for periodic disc shedding by phagocytosis. Only selected proteins in the outer segment are depicted in the inset. N: nucleus


16




Figure 18 Thermally unstable pigment contrasted with bistable/photoreversible pigment 
Left: Cone pigments and rhodopsin are thermally unstable upon activation. The metarhodopsin photoproduct absorbs in the UV, so that it is colorless to human vision and hence the pigment is said to ‘bleach’ in the light. This metarhodopsin decays fairly rapidly, releasing all-trans retinal; cone metarhodopsin II decays in a matter of seconds, and rod metarhodopsin decays in minutes. Right: R-opsins and many C-opsins (other than vertebrate visual opsins) are bistable. The activated all-trans metarhodopsin is photoreversible; it absorbs in the visible part of the spectrum, and upon absorption of a photon is isomerized back to the 11-cis isomer. From Terakita et al (2012). 19

In R-opsins, the photo-activated metarhodopsin is thermally stable (Figure 18 right), with a half-life usually of hours or even days. This active form is rapidly inactivated by the binding of an arrestin molecule, but it remains stably in its all-trans configuration. In most cases this metarhodopsin has its peak absorption in the visible region of the spectrum, indicating that, in its enzymatically active configuration, the Schiff base bond of the all-trans retinaldehyde remains protonated. Furthermore, upon absorption of a further photon, this stable all-trans metarhodopsin (even when arrestin-bound) can undergo photoreversal back to its 11-cis rhodopsin form. Indeed, for most practical purposes this photoreversal is the only short-term mechanism available for the regeneration of visual pigment in many microvillar (rhabdomeric) photoreceptors.

In contrast, the photo-activated metarhodopsin II state of cone and rod opsins is thermally unstable (Figure 18, left), decaying with a half-life that is short (seconds) in cone opsins and somewhat longer (minutes) in rhodopsin (for values, see Table 2 of Imai et al (92)). Fast inactivation occurs as a result of arrestin binding, enabled by rapid phosphorylation. The active meta II absorbs in the UV (~380 nm), because the Schiff base is now un-protonated. Although the protonated meta I intermediate can undergo photoreversal to the 11-cis configuration, the active meta II state is incapable of undergoing such photoreversal, even if it absorbs a blue/UV photon (see Figure 7 of Ritter et al (93)), and this inability is apparently a consequence of an internal molecular rearrangement that accompanies activation.


Absorption of a photon by a rhodopsin or cone-opsin pigment induces isomerization of its retinaldehyde chromophore, activating the receptor. After a brief period, all-trans-retinaldehyde (all-trans-RAL) dissociates from the bleached pigment, rendering it insensitive to light. Before light-sensitivity can be restored, the all-trans-RAL must be reisomerized to 11-cis-retinaldehyde (11-cis-RAL), which recombines with apo-opsin to form a new pigment molecule. Conversion of all-trans-RAL to 11-cis-RAL is performed by a multistep enzyme pathway called the visual cycle. 

The rod and cone photoreceptor cells of the retina utilize a highly photosensitive vitamin A analog, 11-cis retinal, to absorb light and initiate the process of signal transduction. The best understood process for converting
vitamin A (all-trans retinol) from the diet to 11-cis retinal in the retina occurs in the retinal pigment epithelium (RPE). The metabolism of vitamin A and cycling of retinoid analogs between the RPE and photoreceptor cells (the visual cycle) is a complex process, involving a number of specialized proteins and enzymes. 6

This photoisomerization is the initial and only light-dependent reaction in the phototransduction cascade that converts light signal into electrical signal in the photoreceptors. Since the opsins lacking 11-cis-RAL chromophore lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’ 15



The enzymatic production of the visual chromophore via canonical retinoid (visual) cycle. 
11-cis-Retinal regeneration is initiated in the photoreceptor by photoisomerization of the chromophore bound to rhodopsin, its subsequent liberation and reduction to all-trans-retinol. In RPE cells, all-trans-retinol is esterified by LRAT prior to isomerization that is accompanied the acyl cleavage of the ester bond. Resulting 11-cis-retinol is then oxidized to the corresponding aldehyde. These metabolic transformations occur in the smooth ER, where key enzymes of the visual cycle are located. The retinoid cycle is completed by transport of 11-cis-retinal back to the photoreceptors, where it conjugates with opsin and thus regenerates visual pigment. Structures of cellular retinol-binding protein 1 (CRBP1), retinoid isomerase (RPE65) and cellular retinaldehyde-binding protein (CRALBP) correspond to PDB accession numbers 5H8T [79], 3KVC [96], and 4CIZ [97], respectively. Generic model of 11-cis-retinol dehydrogenases (11-cis-RDHs) was built based on the structure of dehydrogenase from Drosophila melanogaster PDB accession number 5ILG. 18



Proteins and parts in the vertebrate visual cycle 

Rhodopsin (also known as visual purple) is a light-sensitive receptor protein involved in visual phototransduction.
Photoreceptor cells are specialized type of cell found in the retina that is capable of visual phototransduction.
Retinal pigment epithelium (RPE) is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells
Retinal G-protein-coupled receptor (RGR) is a non-visual opsin expressed in RPE. RGR bound to all-trans-RAL is capable of operating as a photoisomerase that generates 11-cis-RAL in the light-dependent manner
Interphotoreceptor retinoid-binding protein (IRBP), an abundant 140 kDa glycoprotein secreted by photoreceptors . The binding of retinoids by IRBP protects them from oxidation and isomerization.
β-Carotene 15,15′-monooxygenase (BCO) in RPE supplies all-trans-RAL to the visual cycle via central cleavage of β-carotene 
Cellular retinaldehyde-binding protein (CRALBP)  binds 11-cis-ROL and 11-cis-RAL
Retinoid isomerase RPE65 (or isomerohydrolase) in the RPE. RPE65 is involved in the all-trans to 11-cis isomerization.

The first enzymatic reaction of the visual cycle is the reduction of all-trans-RAL to all-trans-ROL by an NADPH-dependent retinol dehydrogenase (RDH)

Disruption of prRDH in mouse did not affect the rate of 11-cis-RAL regeneration after short light exposure (Maeda et al. 2005). However, it caused significant accumulation of all-trans-RAL following exposure to bright lights and delayed recovery of rod function.
All-trans-ROL is then released from photoreceptors to interphotoreceptor space, where it binds the interphotoreceptor retinoid-binding protein (IRBP), an abundant 140 kDa glycoprotein secreted by photoreceptors . The binding of retinoids by IRBP protects them from oxidation and isomerization. IRBP functions as a transporter protein for all-trans-ROL and 11-cis-RAL during translocation between photoreceptors and RPE cells.
Retinol acyltransferase (LRAT) is the dominant ester synthase responsible for esterification of all-trans-ROL
A second ester synthase called acyl CoA : retinol acyltransferase may complement LRAT to provide additional RE synthase activity under high concentration of all-trans-RO
All-trans-RE was shown to be the substrate for retinoid isomerase RPE65 (or isomerohydrolase) in the RPE. RPE65 is involved in the all-trans to 11-cis isomerization.
Oxidation of 11-cis-ROL to 11-cis-RAL in the RPE is catalysed by RDH5
RPE cells express two retinoid-binding proteins involved in the visual cycle. One is the cellular retinol-binding protein that binds all-trans-ROL, and the other is cellular retinaldehyde-binding protein (CRALBP) that binds 11-cis-ROL and 11-cis-RAL


Vitamin A is converted by the protein RPE65 within the retinal pigment epithelium into 11-cis-retinal.

The retinal pigment epithelium (RPE) closely interacts with photoreceptors in the maintenance of visual function. 3  Without RPE cells, our vision would not be sustainable.   The RPE expresses key metabolic enzymes required for production of the visual chromophore, 11-cis-retinal, and thus comprises an integral part of the retinoid cycle 10

In other words, there is interdependence between the two cell types





Retinoid (visual) cycle. 
Enzymes (red) and binding proteins (blue) involved in 11-cis-retinal regeneration are found in both photoreceptor and RPE cells. Metabolic transformations occurring in the RPE take place in the smooth ER, where key enzymes of the visual cycle are located. PC, phosphotidylcholine. 10



The retinoid cycle regenerates 11-cis-retinal
In rod outer segments (ROS), 11-cis-retinal (11-cis-Ral) couples to a protein opsin, forming rhodopsin. Absorption of a photon of light by rhodopsin causes photoisomerization of 11-cis-Ral to all-trans-retinal (at-Ral) leading to its release from the chromophore-binding pocket of opsin. The movement of at-Ral and certain at-Ral conjugates from the intradiscal face to the cytosolic face of disk membranes is accomplished by the ABC transporter ABCR (also known as ABCA4). At-Ral then is reduced to all-trans-retinol (at-Rol) in a reversible reaction catalyzed by an NADPH-dependent all-trans-retinol dehydrogenase (RDH). At-Rol diffuses across the interphotoreceptor matrix (IPM) facilitated by the interphotoreceptor retinoid-binding protein (IRBP) into the retinal pigment epithelium (RPE) where it is esterified in a reaction catalyzed by lecithin:retinol acyltransferase (LRAT). There, all-trans-retinyl esters may be stored in retinyl ester storage particles (RESTs), also known as retinosomes, or may serve as the substrate for RPE65 that converts them to 11-cis-retinol (11-cis-Rol), which is further oxidized back to 11-cis-Ral by RDH5, RDH11 and other RDHs. 11-cis-Ral formed in the RPE diffuses back into the rod and cone outer segments, where it completes the cycle by recombining with opsins to form rhodopsin and cone pigments. Diseases that result from mutations in proteins involved in the retinoid cycle are indicated in blue boxes. AMD – age-related macular degeneration, CSNB – congenital stationary night blindness, LCA – Leber congenital amaurosis, RP – retinitis pigmentosa. Reproduced with permission from Trends in Biochemical Sciences from reference

The RPE takes up nutrients such as glucose, retinol, and fatty acids from the blood and delivers these nutrients to photoreceptors. Importantly, retinal is constantly exchanged between photoreceptors and the RPE

This molecule is then transported into the photoreceptor cells of the retina, where it acts as a light-activated molecular switch within opsin proteins that activates a complex cascade called the visual cycle. This cycle begins with 11-cis retinal absorbing light and isomerizing into all-trans retinal. The change in shape of the molecule after absorbing light in turn changes the configuration of the complex protein rhodopsin, the visual pigment used in low light levels. This represents the first step of the visual cycle.

Retinal pigment epithelium-specific 65 kDa protein, also known as retinoid isomerohydrolase, is an enzyme of the vertebrate visual cycle 2 RPE65 is expressed in the retinal pigment epithelium(RPE, a layer of epithelial cells that nourish the photoreceptor cells

The isomerohydrolase activity of RPE65 requires coexpression of lecithin retinol acyltransferase in the same cell to provide its substrate. 4

Lecithin retinol acyltransferase is a microsomal enzyme that catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester during phototransduction, an essential reaction for the retinoid cycle in visual system and vitamin A status in liver. 5

Visual cycle
The visual cycle fulfills an essential task of maintaining visual function and needs therefore to be adapted to different visual needs such as vision in darkness or lightness. For this, functional aspects come into play: the storage of retinal and the adaption of the reaction speed. Basically vision at low light intensities requires a lower turn-over rate of the visual cycle whereas during light the turn-over rate is much higher. In the transition from darkness to light suddenly, large amount of 11-cis retinal is required. This comes not directly from the visual cycle but from several retinal pools of retinal binding proteins which are connected to each other by the transportation and reaction steps of the visual cycle.

VISUAL CYCLE OR RETINOID CYCLE


A. Exchange of Retinal Between Photoreceptors and RPE: Isomerization and Reisomerization Between 11-cis and all-trans


Photoreceptors lack cis-trans isomerase function for retinal and are unable to regenerate all-trans-retinal into 11-cis-retinal after transduction of light energy into electrical impulses (30). Therefore, retinal is converted by two major metabolic pathways. One pathway includes binding of retinal to opsin as 11-cis-retinal and release from opsin as all-trans-retinal during recovery of rhodopsin after light absorption. The second pathway serves for the regeneration or better reisomerization of all-trans-retinal into 11-cis-retinal (Fig. 4). The reisomerization occurs in the RPE. Thus retinal entering the second pathway is cycled between photoreceptors and the RPE to ensure constant excitability of photoreceptors. This shows how closely photoreceptors and RPE interact in the process of vision. However, recent studies have described a second pathway of retinal reisomerization (28386). It appears that cones can regenerate a part of their photoisomerized retinal in a second visual cycle occurring in Müller cells. The metabolic pathways of the visual cycle are not completely understood. Most of the understanding comes from studies on mechanisms of retinal degenerations. Much in the following description of the metabolic pathway of retinal regeneration will need to be further verified by extensive future studies.


Restoration of Photoactive Visual Pigments: the Retinoid (Visual) Cycle 9

Equally important to phototransduction is the mechanism that allows resetting to dark conditions after light stimulation. Rhodopsin and other visual pigments are first inactivated by the action of rhodopsin kinase and arrestin (its different spliced forms), but eventually photoactivated rhodopsin needs to be returned to its inactive dark state. The all-trans-retinal released from photoactivated rhodopsin must be converted back to 11-cis-retinal. This metabolic transformation, termed the retinoid (visual) cycle, occurs in the photoreceptor cells and the adjacent RPE (Fig. below).  




The visual (retinoid) cycle. This metabolic renewal of 11-cis-retinal takes place in photoreceptor outer segments and the RPE. First, all-trans-retinal is hydrolyzed from opsin and all-trans-retinal diffuses to the cytoplasmic side where it is reduced to an alcohol by membrane associated 

- all-trans-retinol RDH
Lack of adequate RDH activity can lead to LCA (or RP); all diseases are depicted in red letters. A fraction of all-trans-retinal is released into the intradiscal side. There all-trans-retinal and phosphatidylethanolamine form a Schiff base and together are transported into the cytoplasmic side via 
- ABCA4. Lack of ABCA4 transport activity is associated with Stargardt disease, whereas polymorphisms in this gene are associated with AMD. Retinol diffuses from the cytoplasm to the RPE where it becomes esterified by LRAT to form fatty acid retinyl esters. Such esters have a propensity to coalescence, thermodynamically driving the transfer from photoreceptor to RPE cells. The esters then serve as substrates for the isomerization reaction catalyzed by the 
65-kDa protein, RPE65. The resulting product, 11-cis-retinol, is oxidized to 11-cis-retinal by the 
- 11-cis-retinol specific RDH5 and dual specificity (cis and trans-retinols) RDHs, including RDH10; 11-cis-retinal diffuses back into the photoreceptor outer segments, a process thermodynamically driven by the formation of stable visual pigments. Essential for transporting and protecting these retinoids are intracellular and extracellular retinoid-binding proteins such as
- CRALBP, CRBP1, IRBP and retinol-binding protein 4 (RBP4). Inactivating mutations in the LRAT and RPE65 genes are causes of childhood blindness because these genes are nonredundant, whereas mutations in RDHs and retinoid-binding proteins have less severe effects but can be associated with RP, cone-rod dystrophy, fundus albipunctatus, fundus albescens or Bothnia dystrophy. Retinoids are retained in the eye as a result of LRAT activity. In the bloodstream, retinoids are bound to RBP4 and then enter the eye by passive transport with the help of 
- STRA6. Mutations in the STRA6 gene cause Matthew-Wood Syndrome, a severe disease that includes obesity and mental retardation, as well as faulty eye development and blindness, indicating the importance of STRA6 for retinol transport into the brain and eye.


- RPE-retinal G protein-coupled receptor which isomerizes all-trans-retinal to 11-cis-retinal following light absorption. 
- IRBP, interstitial retinal binding protein; 
- CRBP, cellular retinol binding protein; 
- CRALBP, cellular retinaldehyde binding protein; 
- atRDH, all-trans-retinol dehydrogenase; 
- 11cRDH, 11-cis-retinol dehydrogenase; 
- LRAT, lecithin retinol acyltransferase; 
- RPE65, RPE specific protein with molecular mass of 65 kDa; 
- RGR, RPE-retinal G protein-coupled receptor; 
- RBP/TTR, retinol-binding protein/transthyretin complex.     7


RPE65 Protein Retinoid isomerohydrolase  
Retinal pigment epithelium
Lecithin retinol acyltransferase

Retinoid re-isomerization in darkness 20
A major difference between vertebrate retinal photopigments and those of invertebrates is that the vertebrate visual opsins release their all-trans retinoid following light activation. The vertebrate retina therefore requires a continual supply of 11-cis retinal, in order to silence the residual activity of the free opsin and at the same time regenerate native rhodopsin for further signalling by light. This is accomplished by two biochemical pathways that operate in darkness



Fig. 27. Retinoid cycles: Overview of the two cycles, and flow in the RPE cycle. 
A, Overview of the flow of retinoid in the two cycles. The areas separated by solid lines represent cellular compartments of a retinal pigment epithelial cell (top), a rod and a cone photoreceptor cell (middle), and a Müller cell (bottom). The ovals surrounding 11-Ral represent rhodopsin (grey) and cone opsins (tricolour). Photoisomerization reactions are shown in red. All other chemical reactions are catalysed by enzymes. Retinoids are chaperoned by retinoid-binding proteins (not shown) during intercellular and intracellular movement. Abbreviations: at-RE, all-trans retinyl esters; at-Ral, all-trans retinal; at-Rol, all-trans retinol (=vitamin A); 11-Ral, 11-cis retinal; 11-Rol, 11-cis retinol. From Saari (2012), © Annual Reviews, with permission. 
B, Flow of retinoid in the RPE cycle. Delivery of 11-cis retinoid is shown by solid arrows, while removal of all-trans retinoid is shown by dashed arrows. Abbreviations: OS, outer segment; IPM, inter-photoreceptor matrix; RPE, retinal pigment epithelium; SER, smooth endoplasmic reticulum. RAL, retinaldehyde; ROL, retinol. For the chemical icons: AL, OL and E denote the aldehyde, alcohol, and ester groups attached to the retinoid hydrocarbon chain. Enzymes (in red): RDH, all-trans retinol dehydrogenase; LRAT; lecithin:retinol acyltransferase; RDH5, 11-cis retinol dehydrogenase. Chaperone proteins (in blue): IRBP, inter-photoreceptor retinol binding protein; CRBP, cellular retinol binding protein; RPE65, retinal pigment epithelium 65 kDa protein, now known to be the isomerohydrolase; CRALBP, cellular retinal binding protein. The ABCA4 transporter is not shown; this rescues the very small proportion of all-trans retinoid that inadvertently reaches the luminal leaflet of the disc membrane, and it has only a minor role in total retinoid cycling. From Lamb and Pugh (2006a). Gene names are given in boxes; see also Table 4.

Intra-retina retinoid cycle

Although most work on retinoid recycling over the past four decades has concentrated on the RPE cycle, the fact that cone photoreceptors pre-date rod photoreceptors raises the distinct possibility that the ancestral retinoid cycle of the vertebrate eye may have been the intra-retina cycle involving the cones and Müller cells, with the RPE cycle having evolved subsequently. The intra-retina cycle appears simpler, in that it involves isomerization of all-trans retinol (vitamin A) directly to 11-cis retinol (bottom of Fig. 27A), via what has been termed ‘isomerase 2’ in Müller cells, without requiring initial esterification followed by an isomerohydrolase reaction. A very recent report has indicated that this second isomerase might be the desaturase, DEGS1 (Kaylor et al., 2013), but the identity of the other enzymes that contribute to this intra-retina cycle are only just beginning to emerge. Unfortunately, as this cycle has been less intensively studied, it has not yet proven possible to investigate its evolutionary roots.

RPE retinoid cycle

Two recent papers have made important advances in determining the origin of the classical RPE retinoid cycle. Albalat (2012) and Poliakov et al. (2012) have shown that this cycle is present only in vertebrates, as cephalochordates and tunicates do not possess the required enzymes, implying that the cycle evolved during the 100 million year interval from ④ to ⑤ in Fig. 1.

Two crucial enzymes in the retinoid cycle are LRAT (lecithin:retinol acyltransferase) and RPE65. LRAT esterifies vitamin A (all-trans retinol) to all-trans retinyl ester, which is the substrate for RPE65, which actually performs the isomerization while at the same time cleaving the ester bond, in a so-called ‘isomerohydrolase’ reaction. This step generates 11-cis retinol which is subsequently oxidized to the aldehyde, 11-cis retinal, and then transported to the retina.

Albalat (2012) undertook an in silico search for the genetic machinery of retinoid processing amongst invertebrates, and he analysed the likely function of the components that he found. He concluded that “genome surveys, phylogenetic reconstructions and structural analyses of invertebrate components similar to those of the vertebrate retinoid cycle – that is, Rdh8, Rdh12, Lrat, Rpe65, Rdh5, Rlbp1, and Rbp3 – did not provide any evolutionary or functional support for the existence of the genetic machinery of the retinoid cycle outside vertebrates”.

Poliakov et al. (2012) extended that approach, by experimentally determining the functional activity of the key enzymes LRAT and RPE65 (and similar molecules) in lamprey and in tunicate. In lamprey (P. marinus), they showed that LRAT and RPE65 are both present and functional, and further that the key sites for RPE65's ability to act an isomerohydrolase are remarkably similar to those of jawed vertebrates. On the other hand, their phylogenetic analyses confirmed that neither RPE65 nor LRAT have orthologs in the cephalochordate (amphioxus) or the tunicate (Ciona intestinalis). They showed than an enzyme previously proposed (and named) as a Ciona ortholog of RPE65 by Takimoto et al. (2006) has no isomerohydrolase activity, confirming the report of Kusakabe et al. (2009), and they showed that it functions instead as a BCMO (β-carotene mono-oxygenase).

Taken together, these results show that both the crucial transition from a BCMO to a true RPE65 isomerohydrolase, and also the origin of LRAT, occurred only in the lineage leading to vertebrates (jawless and jawed), after the divergence of tunicates. Hence, the RPE retinoid cycle evolved only in vertebrates.

In a number of invertebrate species, it has been known for many years that rhodopsin can be regenerated in darkness (Stavenga, 1975), but there is scant evidence as to how this occurs. In Drosophila, evidence has recently been obtained for the likely existence of a retinoid cycle, though rather different from that in the RPE (Wang et al., 2010).

Scenario of the origin of retinoid recycling in the vertebrate retina/RPE


From our current knowledge of the intra-retinal and RPE retinoid cycles, it is possible to hypothesize the following scenario for the evolution of retinoid processing in the vertebrate eye:
J-1)
At around the time that chordate ciliary opsins lost their bistable (photoreversible) properties and instead released their all-trans retinoid, an intra-retina retinoid processing cycle arose, that utilized an isomerase (currently termed isomerase 2) in the Müller cells to isomerize retinol from its all-trans to its 11-cis form.
J-2)
Subsequently, around the time that the RPE became specialized as a monolayer apposed to the retina, the two crucial enzymes LRAT and RPE65 evolved, and the RPE was able to adopt an additional role in retinoid re-isomerization.


Retinol Dehydrogenases (RDHs) in the Visual Cycle 1

The isomerization of 11-cis retinal to all-trans retinal in photoreceptors is the first step in vision. For photoreceptors to function in constant light, the all-trans retinal must be converted back to 11-cis retinal via the enzymatic steps of the visual cycle. Within this cycle, all-trans retinal is reduced to all-trans retinol in photoreceptors and transported to the Retinal pigment epithelium (RPE). In the RPE, all-trans retinol is converted to 11-cis retinol, and in the final enzymatic step, 11-cis retinol is oxidized to 11-cis retinal. The first and last steps of the classical visual cycle are reduction and oxidation reactions, respectively, that utilize retinol dehydrogenase (RDH) enzymes. The visual cycle RDHs have been extensively studied, but because multiple RDHs are capable of catalyzing each step, the exact RDHs responsible for each reaction remain unknown. Within rods, RDH8 is largely responsible for the reduction of all-trans retinal with possible assistance from RDH12. retSDR1 is thought to reduce all-trans retinal in cones. In the RPE, the oxidation of 11-cis retinol is carried out by RDH5 with possible help from RDH11 and RDH10. Here, we review the characteristics of each RDH in vitro and the findings from knockout models that suggest the roles for each in the visual cycle.






Crystal structures of proteins involved in the visual cycle, retinoid transport and phototransduction 21
A variety of protein folds are utilized in nature to bind retinoids for metabolism, transport and signal transduction. In panels A-F and H, β strands and α helices are colored blue and green, respectively. 
A) Apocarotenoid oxygenase (ACO) from Synechocystis (PDB ID: 2BIW). 
B) 65 kDa retinal pigment epithelium-specific protein (RPE65, retinoid isomerase) from Bos taurus (PDB ID: 3FSN). The arrow indicates an insertion found in vertebrate members of the carotenoid cleavage enzyme family but not in cyanobacterial members. Despite the overall similar architecture of A) and B), the proteins catalyze fundamentally different reactions and have only about 22% sequence identity. 
C) Human serum retinol-binding protein (PDB ID: 1RBP). 
D) Cellular retinol-binding protein from Rattus norvegicus (PDB ID: 1CRP). 
E) Module two of Xenopus laevis interphotoreceptor retinoid-binding protein (PDB ID: 1J7X). 
F) Human cellular retinaldehyde-binding protein (PDB ID: 3HY5). Proteins that preferentially bind all-trans-retinol (C and D) have retinoid binding sites composed exclusively of β strands whereas those proteins that bind 11-cis-retinal (E and F) are composed of a mixture of α helices and β sheets. 
G) Structural superpositioning of the C-terminal domain of CRALBP and the B domain of IRBP module 2 reveals similar chain folds. Superimposed structures of the C-terminal domain of CRALBP consisting of residues 132-306 (in blue) and the B domain of IRBP module two consisting of residues 89-169, 194-240 and 275-303 (in green) are shown. The bound 11-cis-retinal ligand in the CRALBP structure is shown as orange sticks. Both domains exhibit asymmetric αβα sandwich folds that superimpose with an RMSD of 3.5 Å over 107 matched Cα positions. This observation might indicate that the 11-cis-retinoid-binding site of IRBP resides in the B domain. The superposition was performed with the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). 
H) Ground-state rhodopsin from Bos taurus (PDB ID: 1U19). The retinylidene binding site is composed entirely of α helices.

11



 Retinoid isomerization and enzymatic oxidative cleavage of carotenoids are catalyzed by a family nonheme iron-containing enzymes named carotenoid cleavage oxygenases. Even though the reaction catalyzed by RPE65 does not represent oxygenase activity, RPE65 belongs to this family.  Kiser, a former graduate student and now a junior faculty member in the Department of Pharmacology at Case Western Reserve University, took up the challenge of crystalizing this protein from native bovine RPE (Fig. 8 ), an immense feat that revealed the mechanism of isomerization catalyzed by this protein. The basic RPE65 structural motif is a seven-bladed β-propeller, with the catalytic Fe2+ located in the center of the protein. Two structures of RPE65 were determined in its lipidated and delipidated forms. Our data strongly support retinoid isomerization due to the Lewis acidity of iron that promotes ester cleavage via alkyl-oxygen bond scission to release the acid and generate a resonance-stabilized carbocation intermediate, with eventual production of the 11-cis-retinol product. Previous biochemical studies of RPE65, combined with a proper structural context helped to reveal the mechanism of isomerization more than any of the studies separately. This area of research is still very active because it concerns the fundamentals of catalytic isomerization. Mutations in RPE65 are known to be responsible for LCA.  Mice lacking this enzyme recapitulate the human condition.  Moreover, animal studies of this mutation were critical in developing several promising therapies.  


Crystal structure of RPE65. 
(A) Cartoon representation showing the beta-propeller fold of RPE65. The catalytic iron, located on the propeller axis, is coordinated by four conserved histidine (His) residues. Three second sphere Glu residues are also important active site components. Residues of Glu and His are shown in stick representations. 
(B) Dimeric structure of RPE65. The hydrophobic patches that mediate RPE65 membrane association (brown) are oriented in parallel. The entrance to the active site cavity (dashed black lines) is surrounded by residues comprising the hydrophobic patch (PDB accession code: 4F2Z I). 

Retinoids need to be shuttled between different organelles and protected from isomerization, oxidation, and condensation. Thus, key retinoid-binding proteins are critical for maintaining proper retinoid isomeric and oxidation states. Cellular retinaldehyde–binding protein (CRALBP) in the RPE and Müller cells, and extracellular interphotoreceptor retinoid–binding protein (IRBP) are two major carriers involved.  The structure of CRALBP—with its unanticipated isomerase activity—has been elucidated, whereas the structure of IRBP has only been partially characterized. Inactivating mutations in either one of these binding proteins can cause retinal degenerative disease.  

Evolution and the origin of the visual retinoid cycle in vertebrates 12

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.

Restoration of light sensitivity requires chemical reisomerization of retinaldehyde via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE).

The closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.

The interphotoreceptor retinoid binding (IRBP) is essential for normal retinoid processing in cone photoreceptors 13 While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways.



Possible scenarios for the chordate visual cycle evolution. Schematic of cellular and subcellular localizations of visual cycle proteins in vertebrates, tunicates and protostomes. The dendrogram represents proposed evolutionary routes from the ancestral state in a bilaterian ancestor to the extant systems. Dashed branches indicate two possible origins of the visual cycle in the neural complex of the adult ascidian. Events occurred during evolution are indicated by purple arrows. Arrows with a question mark indicate alternative possibilities for the events. OS, outer segments of photoreceptor cells.



Schematics of two visual cycles in vertebrate eye.
The canonical RPE visual cycle (left) recycles all-trans-retinol (at ROL) released from rods and cones following a bleach to 11-cis-retinal (11c ROL), which can be used by both rods and cones for pigment regeneration. The retina visual cycle (right) relies on the Müller cells to recycle all-trans-retinol released from cones to 11-cis-retinol, which only cones can move to their outer segments and oxidize to 11-cis-retinal for regeneration of the pigment. IPM, interphotoreceptor matrix. hν, photon of light. 17


1. https://en.wikipedia.org/wiki/Retinol#Vision
2. http://www.wikiwand.com/en/RPE65
3. http://physrev.physiology.org/content/85/3/845
4. http://www.pnas.org/content/102/35/12413.full
5. https://en.wikipedia.org/wiki/Lecithin_retinol_acyltransferase
6. http://sci-hub.cc/http://www.sciencedirect.com/science/article/pii/S135094620300051X
7. http://physrev.physiology.org/content/85/3/845
8. http://sci-hub.cc/http://www.sciencedirect.com/science/article/pii/B9780123742032001858
9. http://iovs.arvojournals.org/article.aspx?articleid=2166602
10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858459/
11. http://sci-hub.cc/http://www.nature.com/nchembio/journal/v11/n6/full/nchembio.1799.html?foxtrotcallback=true
12. http://rstb.royalsocietypublishing.org/content/364/1531/2897
13. https://www.ncbi.nlm.nih.gov/pubmed/20238012
14. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680234/
15. http://rstb.royalsocietypublishing.org/content/364/1531/2897
16. http://photobiology.info/Terakita.html
17. http://www.jbc.org/content/287/3/1635.full
18. http://www.mdpi.com/2072-6643/8/11/676/htm
19. https://www.ncbi.nlm.nih.gov/books/NBK153508/
20. http://www.sciencedirect.com/science/article/pii/S1350946213000402#bib142
21. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891588/
22. www.mdpi.com/2072-6643/8/11/746/pdf

Structural approaches to understanding retinal proteins needed for vision
[size=13]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971393/



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Type-I opsins common to bacteria and other taxa are not homologous to animal opsins

Two types of ion channel delineate different modes of phototransduction known in animals. First, 

1.  Cyclic nucleotide–gated (CNG)  ion channels function in a ciliary phototransduction cascade, whereby CNG modulation effects a hyperpolarizing potential in the photoreceptor cell's response to light.


Cyclic nucleotide–gated ion channels or CNG channels are ion channels that function in response to the binding of cyclic nucleotide (cNMP). 

cyclic nucleotide (cNMP) is a single-phosphate nucleotide with a cyclic bond arrangement between the sugar and phosphate groups. Like other nucleotides, cyclic nucleotides are composed of three functional groups: a sugar, a nitrogenous base, and a single phosphate group. As can be seen in the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) images, the 'cyclic' portion consists of two bonds between the phosphate group and the 3' and 5' hydroxyl groups of the sugar, very often a ribose.




Cyclic adenosine monophosphate. The cyclic portion refers to the two single bonds between the phosphate group and the ribose


Cyclic nucleotides are produced from the generic reaction NTP → cNMP + PPi, where N represents a nitrogenous base. The reaction is catalyzed by specific nucleotidyl cyclases, such that production of cAMP is catalyzed by adenylyl cyclase and production of cGMP is catalyzed by guanylyl cyclase    Adenylyl cyclase has been found in both a transmembrane and cytosolic form, representing distinct protein classes and different sources of cAMP.


CNG channels are nonselective cation channels that are found in the membranes of various tissue and cell types, and are significant in sensory transduction as well as cellular development. Their function can be the result of a combination of the binding of cyclic nucleotides (cGMP and cAMP) and either a depolarization or a hyperpolarization event. Initially discovered in the cells that make up the retina of the eye, CNG channels have been found in many different cell types across both the animal and the plant kingdoms. CNG channels have a very complex structure with various subunits and domains that play a critical role in their function. CNG channels are significant in the function of various sensory pathways including vision and olfaction, as well as in other key cellular functions such as hormone release and chemotaxis. CNG channels have also been found to exist in prokaryotes, including a large number of spirochaeta, though their precise role in bacterial physiology remains unknown.

2. Conversely, canonical transient receptor potential (TRPC) ion channels, members of a larger gene family of TRP channels , function in a rhabdomeric pathway where activation leads to a depolarizing cell-physiological response to light

Both CNG- and TRPC-modulated pathways are initiated by class-specific opsin paralogues (c-opsin and r-opsin, respectively), which are both present in protostomes and deuterostomes, and therefore predate the origin of bilaterian animals. Despite the centrality of these signalling pathways to vision and other photosensitivity phenotypes, an understanding of the origins of these cascades in animal evolution has evaded biologists, largely because accumulation of data from non-bilaterian animals like Cnidaria has lagged far behind the availability of data from bilaterians—especially flies, molluscs and vertebrates. Despite the centrality of these signalling pathways to vision and other photosensitivity phenotypes, an understanding of the origins of these cascades in animal evolution has evaded biologists

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8 Retinal on Tue Oct 17, 2017 6:29 am

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Retinal

11-cis-retinal. 11-cis-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  Retinal is a polyene organic compound, meaning that this molecule contains multiple alkene functional groups. 4 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 cis to trans configuration change. This configuration change causes a change in the conformation (the three-dimensional shape) of the opsin protein.





One of the basic and unresolved puzzles in the chemistry of vision concerns the natural selection of 11-cis-retinal as the light-sensing chromophore in visual pigments. 3 The results show that the electrostatic interaction between retinal and opsin dominates the natural selection of 11-cis-retinal over other cis isomers in the dark state.



Electron redistribution resulting from photon absorption could be responsible for the bond rotation and rearrangement of amino acid residues leading to protein activation. 7  If allowed to occur, the subsequent chromophore shape change due to isomerization completes proton pumping in some cases (class 1 and apparently some putative class 2 rhodopsins), removes the antagonistic activity of the β-ionone moiety toward completion of rhodopsin activation in other cases, and may influence later steps in the visual photocycle. We further found that the chromophore-binding site is highly sensitive to the charge separation in the chromophore. The large shift in the action spectra of the two-naphthaldehyde series in Chlamydomonas and the absorption spectrum for bacteriorhodopsin shows interaction of the excitation of these chromophores with the nearby polarizable amino acids. Hence, the light-induced consequences of charge separation are necessary and sufficient to initiate the phototransduction process. 


Visual transduction is the process by which visual retina cells convert light into a neural signal, which is in turn transmitted to the brain along the optic nerve. It is initiated by photoisomerization of 11-cis-retinal  to all-trans-retinal upon light absorption. This event gives rise to a series of conformational changes in the chromophore, linked to changes in the protein structure (such as displacement of helices and reorganization of cytoplasmic loops), deprotonation of the Schiff base (with concomitant proton uptake by Glu134), and hydrolysis of trans-retinal from the apoprotein. 5



The considerable reduction in regeneration and efficiency with these ringdemethylated retinal analogues, a likely indication of slow equilibrium between binding and unbinding, together with the results for the acyclic analogues, demonstrated the importance of the ring methyl groups in anchoring the chromophore in the binding site as an inverse agonist.


Synthesis of the All-trans-retinal Chromophore of Retinal G Protein-coupled Receptor Opsin 6
Carotenoids are C40 isoprenoids which consist of eight isoprene units and can be divided in two major groups: carotene and xanthophylls. 5

We are all familiar with carotenoids as the yellow to red coloring of fruits, flowers and vegetables. These colored compounds, C40 isoprenoids, are synthesized in plants, certain fungi and bacteria. A carotenoid is the precursor of vitamin A by  β-carotene conversion in the small intestine of mammals. For this reaction, a central cleavage mechanism at the C-15,C-15′ double bond for the conversion of β-carotene to vitamin A is required. The β-carotene-cleavage enzyme depended on molecular oxygen and thus the enzyme was termed β,β-carotene-15,15′-oxygenase (BCO). Oxidative cleavage at the central (15,15′) double bond is catalyzed in a monooxygenase mechanism via a transient carotene epoxide.  

All animals endowed with the ability to detect light through visual pigments must have evolved pathways in which dietary precursors for chromophore, such as carotenoids and retinoids, are first absorbed in the gut, and then transported, metabolized and stored within the body to establish and sustain vision. Carotenoids and their retinoid metabolites are isoprenoid compounds, which physiologically undergo only a limited number of possible chemical transformations.The process can be divided into three major events: absorption of precursors in the gut, transport and storage in the body, and uptake into cells that produce chromophore.

Two fundamental processes in chromophore metabolism defied molecular analysis for a long time: the conversion of the parent C40 carotenoid precursor into C20 retinoids and the all-trans to 11-cis isomerization and cleavage involved in continuous chromophore renewal. 

retinal pigment epithelial (RPE)
Lecithin-retinol acyltransferase
Retinyl ester hydrolase
11-cis-retinol dehydrogenases
Isomerohydrolase
Retinoid-binding proteins
RPE retinal G protein-coupled receptor (RGR)

The retinal pigment epithelial (RPE) cells are highly active in the metabolism of retinoids and are essential for the synthesis of the 11-cis-retinal chromophore of visual pigments  Many specialized enzymes and retinoid-binding proteins are involved in the production of 11-cis-retinal from all-trans-retinol.
Lecithin-retinol acyltransferase
is among the most active enzymes in retinoid processing and acts early in the retinoid cycle by catalyzing the esterification of all-trans-retinol soon after uptake into the RPE cells

Other enzymes that affect the content and distribution of retinoids include retinyl ester hydrolase.  Lecithin retinol acyltransferase is a microsomal enzyme that catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester during phototransduction, an essential reaction for the retinoid cycle in visual system and vitamin A status in liver. 

The biochemical and structural basis for trans-to-cis isomerization of retinoids in the chemistry of vision 8

There is an intriguing evolutionary conservation of the key components involved in chromophore production and recycling, these genes also have adapted to the specific requirements of both insect and vertebrate vision. Visual GPCR signaling is unique with respect to its dependence on a diet-derived chromophore (retinal or 2-dehydro-retinal in vertebrates; retinal and 3-hydroxy-retinal in insects). The chromophore is naturally generated by oxidative cleavage of carotenoids (C40) to retinoids.(C20). Then the retinoid cleavage product must be metabolically converted to the respective 11-cis-retinal derivative in either the same carotenoid cleavage reaction or a separate reaction. All animals endowed with the ability to detect light through visual pigments must have evolved pathways in which dietary precursors for chromophore, such as carotenoids and retinoids, are first absorbed in the gut, and then transported, metabolized and stored within the body to establish and sustain vision. The 11-cis-stereoisomer binds by a Schiff-base linkage to a membrane-embedded Lys residue in opsin to form functional visual pigments.

11-cis-retinol dehydrogenases, and a putative isomerohydrolase or isomerase;
The retinoid-binding proteins in the RPE include cellular retinol-binding protein, cellular retinaldehyde-binding protein, and a unique opsin, RPE retinal G protein-coupled receptor (RGR)
RGR is involved in the formation of 11-cis-retinal  and is necessary for maintaining normal steady-state levels of both 11-cis-retinal and rhodopsin in a light-adapted eye


Key enzymatic steps in carotenoid/retinoid metabolism in insects and mammals
A comparison of the chemical transformations of carotenoids and their retinoid metabolites in the pathways for chromophore production in different animal classes. These include oxidative cleavage of double bonds, oxidation of alcohols to aldehydes and aldehydes to acids and aldehyde reduction to alcohols, esterification of alcohols, hydroxylation of carbons in ionone ring structures, and trans-to-cis isomerization of carbon-carbon double bonds. A) In insects, carotenoids such as zeaxanthin are converted to one molecule of 11-cis and one molecule of all-trans-3-hydroxy-retinal in an isomerooxygenase reaction. 
[ii] all-trans-3-hydroxy-Retinal is converted to all-trans-3-hydroxy-retinol. 
[iii] all-trans-3-hydroxy-Retinol is light-dependently isomerized to 11-cis-3-hydroxy-retinol. 
[iv] 11-cis-3-hydroxy-Retinol is oxidized to 11-cis-3-hydroxy-retinal. B) In mammals, β,β-carotene is symmetrically cleaved to two molecules of all-trans-retinal. [ii] all-trans-Retinal is reduced to all-trans-retinol (vitamin A). [iii] all-trans-Retinol is converted to retinyl esters for storage or [iv] formation of 11-cis-retinol. [v] 11-cis-Retinol is oxidized to 11-cis-retinal. [vi] all-trans-Retinal can be also oxidized to retinoic acid.


Carotenoids biosynthesis in Chlamydomonas reinhardtii





Schematic diagram of the carotenoid biosynthetic pathway in plants and microalgae. 6

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

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891588/
2. http://www.sciencedirect.com/science/article/pii/S0042698906003580
3. https://sci-hub.bz/http://pubs.acs.org/doi/pdf/10.1021/ja208789h
4. http://www.dummies.com/education/science/chemistry/how-conjugated-double-bond-stereochemistry-works-in-vision/
5. http://chlamypw.mpimp-golm.mpg.de/CHLAMY/new-image?object=CAROTENOID-PWY&type=PATHWAY
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125507/
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400536/
8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891588/

Spectral properties and isomerisation path of retinal in C1C2 channelrhodopsin
http://pubs.rsc.org/en/content/articlehtml/2015/cp/c5cp02650d



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9 Cone and Rod photoreceptor cells on Tue Oct 17, 2017 3:19 pm

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Cone and Rod photoreceptor cells

In the retinas of most vertebrates, there are two types of photoreceptor cells, rods and cones (Fig. 1). Rods are responsible for scotopic vision, the vision working under dim light conditions where cones are not functional, whereas photopic vision, the vision working under daylight conditions is mediated by cones. In agreement with this visual duplicity, rods are more sensitive than cones and can generate a response from even a single photon. Although less sensitive than rods, cones respond and regenerate more rapidly than rods and exhibit considerably greater adaptive ability than rods. Rods contain a single rod visual pigment (rhodopsin), whereas cones use several types of cone visual pigments with different absorption maxima. Integration of the photon signals from cones having cone visual pigments with different absorption maxima enables animals to discriminate the color of materials. 1

The two kinds of photoreceptors in the retinas of vertebrates—the rods and cones—differ in many ways, both anatomically and functionally. The main difference is the opposite roles that they play in vision. The rods provide what is called scotopic vision: they are very sensitive to low levels of light but cannot distinguish colours. The cones provide photopic vision: they require bright light but let us see the world around us in colour, and more sharply.

In both cases, however, the neural response is the same—the hyperpolarization of the photoreceptor cells—and is initiated by the same phenomenon: the absorption of light energy by photopigments embedded in the discs of the photoreceptors' outer segments. In the rods, the photosensitive pigment is rhodopsin, which has its peak sensitivity at around 500 nanometres (nm) in the visible-light band of the electromagnetic spectrum.

In the cones, the photosensitive pigment is opsin, a transmembrane protein that is very similar to rhodopsin. Opsin comes in three different varieties, distinguished by differences in their amino acid sequences that result in differences in their light-absorption curves, with peaks in the blue, green, and red portions of the visible light spectrum, respectively.

All three varieties of opsin are present in all cones. But there are three types of cones, in each of which a different variety of opsin heavily predominates, making it more sensitive to a different part of the colour spectrum, as shown in the diagram here. "Blue" cones containing mostly blue-sensitive opsin are excited chiefly by a wavelength of around 420 nm, "green" cones by a wavelength around 530 nm, and "red" cones by a wavelength near 560 nm. 2

1. http://www.sciencedirect.com/science/article/pii/S0005272813001461
2. http://thebrain.mcgill.ca/flash/a/a_02/a_02_m/a_02_m_vis/a_02_m_vis.html

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The irreducible process of phototransduction, 11 cis retinal synthesis, and the visual cycle, essential for vertebrate vision

http://reasonandscience.heavenforum.org/t1638-origin-of-phototransduction-the-visual-cycle-photoreceptors-and-retina#5753

William Bialek: More Perfect Than We Imagined - March 23, 2013
Excerpt: photoreceptor cells that carpet the retinal tissue of the eye and respond to light, are not just good or great or phabulous at their job. They are not merely exceptionally impressive by the standards of biology, with whatever slop and wiggle room the animate category implies. Photoreceptors operate at the outermost boundary allowed by the laws of physics, which means they are as good as they can be, period. Each one is designed to detect and respond to single photons of light — the smallest possible packages in which light comes wrapped. “Light is quantized, and you can’t count half a photon,” said William Bialek, a professor of physics and integrative genomics at Princeton University. “This is as far as it goes.” … In each instance, biophysicists have calculated, the system couldn’t get faster, more sensitive or more efficient without first relocating to an alternate universe with alternate physical constants. 9

From the book: Evolution of Visual and Non-visual Pigments, page 106
Opsin—the protein that underlies all animal vision., has become a favorite research target, not only of vision scientists but of many researchers interested in the evolution of protein structure, function, and specialization. This level of focus has made the opsins canonical G-protein-coupled receptors (GPCRs) and arguably the most investigated protein group for its evolutionary radiations and diverse functional specializations.  Still, opsin’s early evolution REMAINS PUZZLING, and there are many questions throughout its evolutionary history for which we have partial, but tantalizingly incomplete, answers. Obviously, the invertebrates, with their astonishing diversity and with evolutionary hints of the most ancient animals in their genomes, functions, and even body plans, offer the best hope of answering many of these fundamental questions.

Rhodopsins and Cone opsins have two interdependent agents, namely 11 cis retinal chromophores, and opsins, to which they are attached. By absorbing a photon, 11 cis retinal isomerizes to trans retinal conformation, and that triggers a conformational change in opsins, which trigger the signal transduction cascade, which in the end, provokes the electrical signal, transmitted to the brain for processing.

11-cis-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

There are many things that are functionally important, and must be JUST RIGHT, in order for these molecular mechanisms to work. 

The fact that rhodopsin has been intensely studied, provides a WEALTH of information on a molecular level, which permits to make INFORMED CONCLUSIONS of its origins.

Now OBSERVE how many things must be JUST RIGHT and ESSENTIAL ( following is straightforward from the relevant scientific literature ) :

Rhodopsin Structure and Activation

Rhodopsin consists of an apoprotein opsin and an inverse agonist ( that's like a mechanism which keeps a switch off ), the 11-cis-retinal chromophore, which is covalently bound through a Schiff base linkage to the side chain of Lys296 of opsin protein.

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

- a Schiff base linkage   
- a Lys296 residue where chromophore retinal covalently binds
- the side chain of the residue
- an essential amino acid residue called "counter ion" key factor appears to be the protonation state of the Schiff-base counterion
- a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of  rhodopsin
A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. 2

Residues important for stabilizing the tertiary structure

- (e.g. disulphide bridge (S-S),
- amino-terminal (N) glycosylation sites)
- activation/deactivation of photopigments (e.g. carboxyl-terminal (C) phosphorylation sites)
- membrane anchorage (e.g. palmitoylation sites)

For visible light absorption, all opsins contain an essential amino acid residue called "counter ion", in addition to a retinal-binding site, Lys296 (in the bovine rhodopsin numbering system), where chromophore retinal covalently binds through a protonated Schiff base linkage . The proton on the Schiff base is necessary for visible light absorption, but energetically unstable within the opsin molecule. In opsin pigments, a negatively charged amino acid residue, counterion, stabilizes the protonated Schiff base, and is an essential amino acid residue for opsin pigments to absorb visible light.

Various types of opsin-based pigments with absorption maxima in the visible light region possess a “protonated” Schiff base linkage. In the protein moiety, the positive charge on the protonated Schiff base is unstable, and therefore a counterion, a negatively charged amino acid residue is needed to stabilize the positive charge. In vertebrate visual pigment, glutamic acid at position 113 serves as the counterion 11

Furthermore: movement of the cytoplasmic end of the sixth transmembrane helix is essential for pigment activation.

From the above information, it is clear that there is an evidently FINE- TUNED protein-protein interaction, that is, the 11 cis retinal chromophore physical constitution, and the opsin physical constitution, MUST BE JUST RIGHT from the beginning, and be able to interact PRECISELY to trigger the signal transduction chain.

Let's suppose, opsin is able to interact with TRANSDUCIN. So what ?? If the signal transduction pathway is not fully setup, and able to go all the way through - no signal - no vision. So having such a precise protein-protein arrangement will make only sense, if down down there, after many complex molecular interactions, a visual image is generated in the brain. After two amplification steps, the goal is achieved, and a signal is sent to the brain. To get that signal, is a REMARKABLE SIGNAL AMPLIFICATION mechanism:

A single photoactivated rhodopsin catalyzes the activation of 500 transducin molecules. Each transducing can stimulate one cGMP phosphodiesterase molecule and each cGMP phosphodiesterase molecule can break down 1000 molecules of cGMP per second. Therefore, a single activated rhodopsin can cause the hydrolysis of more than 100.000 molecules of cGMP per second.

Following enzymes, molecules, and proteins are ESSENTIAL in the signal transduction pathway:

Rhodopsin  Rhodopsin is an essential G-protein coupled receptor in phototransduction.
Retinal Schiff base cofactor All-trans-retinal is also an essential component of type I, or microbial, opsins such as bacteriorhodopsinchannelrhodopsin, and halorhodopsin.
Transducin  Their function is to mediate the signal transduction from the photoreceptor proteins, the opsins, to the effector proteins, the phosphodiesterases 6
Guanosine diphosphate ( GDP ) Transducin is tightly bound to a small organic molecule called Guanosine diphosphate ( GDP ) 
Guanosine triphosphate GTP when it binds to rhodopsin the GDP dissociates itself from transducin ana molecule called  GTP, which is closely related to, but critically different from, GDP, binds to transducin. 
G-nucleotide exchange factor (GEF)    The exchange of GDP for GTP is done by a G-nucleotide exchange factor (GEF) 7
Cyclic guanosine monophosphate (cGMP) 
phosphodiesterase (PDE)  is necessary to transform cGMP to GMP. This closes the cGMP gated ion channel due to the decreasing amounts of cGMP in the cytoplasm 6  
cGMP-gated channel of rod photoreceptors
Cyclic nucleotide-gated Na+ ion channels

Once the signal goes through,  a system is required to stop the signal that is generated and restore the opsin to its original state. For that task, other essential proteins are needed  to restore the initial state of rhodopsin:

Guanylate cyclase
Rhodopsin kinase
Arrestin

The biosynthesis of 11 Cis retinal, essential in the first step of vertebrate vision, is also REMARKABLE.

There is an INTRIGUING EVOLUTIONARY CONSERVATION  of the key components involved in chromophore production and recycling, these genes also have adapted to the specific requirements of both insect and vertebrate vision. Visual GPCR signaling is unique with respect to its dependence on a diet-derived chromophore (retinal or 2-dehydro-retinal in vertebrates; retinal and 3-hydroxy-retinal in insects). The chromophore is naturally generated by oxidative cleavage of carotenoids (C40) to retinoids.(C20). Then the retinoid cleavage product must be metabolically converted to the respective 11-cis-retinal derivative in either the same carotenoid cleavage reaction or a separate reaction. 3

All animals endowed with the ability to detect light through visual pigments need pathways in which dietary precursors for chromophore, such as carotenoids and retinoids, are first absorbed in the gut, and then transported, metabolized and stored within the body to establish and sustain vision.

Two fundamental processes in chromophore metabolism defied molecular analysis for a long time: the conversion of the parent C40 carotenoid precursor into C20 retinoids and the all-trans to 11-cis isomerization and cleavage involved in continuous chromophore renewal. Following proteins are essential in the pathway to synthesize 11 cis retinals :

retinal pigment epithelial (RPE)  The retinal pigment epithelium (RPE), a single layer of cuboidal cells lying betweenBruch's membrane and the photoreceptors, is an essential component of the visual system.
Lecithin-retinol acyltransferase  Is Essential for Accumulation of All-trans-Retinyl Esters in the Eye and in the Liver 4
Retinyl ester hydrolase
11-cis-retinol dehydrogenases
Isomerohydrolase  It performs the essential enzymatic isomerization step in the synthesis of 11-cis retinal. 5
Retinoid-binding proteins
RPE retinal G protein-coupled receptor (RGR)

The absorption of light by rhodopsin results in the isomerization of the 11- cis -retinal chromophore to all- trans forming the enzymatically active intermediate, metarhodopsin II, which commences the visual transduction process.

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.

Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. 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.

Restoration of light sensitivity requires chemical reisomerization of trans-retinal via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE).

When a photon of light is absorbed, 11-cis retinal is transformed to all-trans retinal, and it moves to the exit site of rhodopsin. It will not leave the opsin protein until another fresh chromophore comes to replace it, except for in the ABCR pathway. Whilst still bound to the opsin, all-trans retinal is transformed into all-trans retinol by all-trans Retinol Dehydrogenase. It then proceeds to the cell membrane of the rod, where it is chaperoned to the Retinal Pigment Epithelium (RPE) by Interphotoreceptor Retinoid Binding Protein (IRBP). It then enters the RPE cells, and is transferred to the Cellular Retinol Binding Protein (CRBP) chaperone. 8

The visual cycle fulfills an essential task of maintaining visual function and needs therefore to be adapted to different visual needs such as vision in darkness or lightness. For this, functional aspects come into play: the storage of retinal and the adaption of the reaction speed. Basically vision at low light intensities requires a lower turn-over rate of the visual cycle whereas during light the turn-over rate is much higher. In the transition from darkness to light suddenly, large amount of 11-cis retinal is required. This comes not directly from the visual cycle but from several retinal pools of retinal binding proteins which are connected to each other by the transportation and reaction steps of the visual cycle.

This cycle is present only in vertebrates, as cephalochordates and tunicates do not possess the required enzymes. The isomerization of 11-cis retinal to all-trans retinal in photoreceptors is the first step in vision. For photoreceptors to function in constant light, the all-trans retinal must be converted back to 11-cis retinal via the enzymatic steps of the visual cycle. Within this cycle, all-trans retinal is reduced to all-trans retinol in photoreceptors and transported to the Retinal pigment epithelium (RPE). In the RPE, all-trans retinol is converted to 11-cis retinol, and in the final enzymatic step, 11-cis retinol is oxidized to 11-cis retinal. The first and last steps of the classical visual cycle are reduction and oxidation reactions, respectively, that utilize retinol dehydrogenase (RDH) enzymes.

To make things even more intriguing, there are at least 4 different pathways for regeneration of 11 Cis retinal. 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. In the vertebrate cycle, following proteins are ESSENTIAL :

Rhodopsin (also known as visual purple) is a light-sensitive receptor protein involved in visual phototransduction.
Photoreceptor cells are specialized type of cell found in the retina that is capable of visual phototransduction.
Retinal pigment epithelium (RPE) is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells
Retinal G-protein-coupled receptor (RGR) is a non-visual opsin expressed in RPE. RGR bound to all-trans-RAL is capable of operating as a photoisomerase that generates 11-cis-RAL in the light-dependent manner
Interphotoreceptor retinoid-binding protein (IRBP), an abundant 140 kDa glycoprotein secreted by photoreceptors . The binding of retinoids by IRBP protects them from oxidation and isomerization.
β-Carotene 15,15′-monooxygenase (BCO) in RPE supplies all-trans-RAL to the visual cycle via central cleavage of β-carotene 
Cellular retinaldehyde-binding protein (CRALBP)  binds 11-cis-ROL and 11-cis-RAL
Retinoid isomerase RPE65 (or isomerohydrolase) in the RPE. RPE65 is involved in the all-trans to 11-cis isomerization.


Retinoids need to be shuttled between different organelles and protected from isomerization, oxidation, and condensation. Thus, key retinoid-binding proteins are critical for maintaining proper retinoid isomeric and oxidation states. Cellular retinaldehyde–binding protein (CRALBP) in the RPE and Müller cells, and extracellular interphotoreceptor retinoid–binding protein (IRBP) are two major carriers involved.  The structure of CRALBP—with its unanticipated isomerase activity—has been elucidated, whereas the structure of IRBP has only been partially characterized. Inactivating mutations in either one of these binding proteins can cause retinal degenerative disease.


Origin of opsins: 

Type I and Type II opsins
Opsins comprise two protein families, called type I and type II opsins, with detailed functional similarities. Both opsin classes are seven-transmembrane (7-TM) proteins that bind to a lightreactive chromophore to mediate a diversity of responses to light. In both families, the chromophore (retinal) binds to the seventh TM domain via a Schiff base linkage to a lysine amino acid.  Two major classes of opsins are defined and differentiated based on primary protein sequence, chromophore chemistry, and signal transduction mechanisms. Several lines of evidence indicate that the two opsin classes evolved separately, illustrating an amazing case of convergent evolution.

Convergent evolution ? Can you believe that ? 

Supposed split of type 1 and type 2 opsins: 
Although parapinopsin ( Any of a group of opsins in the parapineal gland of some fish )  has an amino acid sequence similar to those of vertebrate visual pigments, it has the molecular properties of a bistable pigment, similar toinvertebrate visual pigments (Gq-coupled visual opsin) and Opn3 (encephalopsin)/ TMT-opsin-based pigments. These observations indicate that parapinopsin is one of the key pigments for understanding the molecular evolution of vertebrate visual pigments. Parapinopsin has glutamic acid residues at both positions 113 and 181, similar therefore to vertebrate visual pigments. However, mutational analyses have revealed that Glu181 is the functional counterion residue, as found for invertebrate rhodopsins. Therefore, this suggests that the molecular properties of photoproducts, namely photoregeneration (bistability) and bleaching, may relate to counterion position and that vertebrate visual pigments having bleaching property might have evolved from an ancestral vertebrate bistable pigment similar to parapinopsin.  

This might be not that easy. In order for the transition to work, all the proteins and enzymes, and all metabolic steps of the visual cycle would have to be set up and in place, fully working, otherwise, how could 11 cis retinal be regenerated? and since there are at least 4 different visual cycles, they would have had to emerge independently four times..... and if key retinoid-binding proteins were not there ready to bind retinoid, nothing done.....

Now - THIS is the kind of information that must be studied, considered, and analyzed when talking about origins of vision and phototransduction. 

What is the proposal based on philosophical naturalism to explain the systems described above ? 

The Evolution of Opsins 
T H Oakley and D C Plachetzki, 2012

Opsin genes were very often duplicated and retained during animal evolution. Early opsin gene duplications led to the major opsin groups and more recent duplications mostly led to additional specializations, such as the ability for color vision. As members of highly coordinated protein networks, changes in opsin proteins are sometimes correlated with changes in partnering proteins. The interaction of two evolutionary processes has resulted in the diversity of opsin-based phototransduction pathways observed today that contains a combination of shared and distinct interactions. First, co-option refers to instances where an opsin recruited different intracellular signaling components than its ancestor during evolution. Second, coduplication involved the simultaneous duplication of multiple genes of an ancestral network. Co-option and coduplication are not discrete alternatives; instead, some genes of a network originated by co-duplication, whereas others joined the network by co-option.

Where is the evidence for these claims? So, basically, they claim duplication and co-option did the feat. And furthermore, they go fishing where they should not, using teleological phrasing, like recruited. Recruiting is a conscient direction driven mental process based on intelligence. There is not a shred of evidence for the proposal, nonetheless, it is presented as consumed, proven fact. This is the bitter fruits of methodological naturalism.


1. http://www.sciencedirect.com/science/article/pii/S0042698906003580
2. http://www.nature.com/nature/journal/v471/n7340/abs/nature09795.html
3. https://www.researchgate.net/publication/41621159_The_biochemical_and_structural_basis_for_trans-to-cis_isomerization_of_retinoids_in_the_chemistry_of_vision
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351249/
5. https://www.ncbi.nlm.nih.gov/gene/6121
6. https://www.researchgate.net/publication/267825362_Evolution_of_transducin_alpha_beta_and_gamma_subunit_gene_families
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794341/
8. https://en.wikipedia.org/wiki/Bleach_and_recycle
9. http://darwins-god.blogspot.com/2013/03/william-bialek-more-perfect-than-we.html

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11 Intracellular rhodopsin transport on Mon Oct 30, 2017 9:13 am

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Intracellular rhodopsin transport

Eyespot placement is dictated by the cytoskeleton, specifically by properties of the D4 rootlet. 1 


The C. reinhardtii eyespot. 
(a) A diagram illustrating asymmetric localization of the eyespot relative to the cytoskeleton. Two flagella and four microtubule rootlets extend from a pair of basal bodies at the anterior end of the cell; both the mother basal body (small black oval) and the daughter basal body (small gray oval) are associated with a four-membered rootlet (M4 or D4) and a two-membered rootlet (M2 or D2). The single eyespot (large oval) is associated with the four-membered daughter rootlet (D4), and the flagellum assembled from the daughter basal body is termed the cis-flagellum, whereas the trans-flagellum is assembled from the mother basal body. 
(b) A light micrograph of a wild-type C. reinhardtii cell showing the single large equatorial eyespot. 
(c) A diagram illustrating the components of the eyespot. Rhodopsin photoreceptors, including ChR1 (light gray ovals), in the plasma membrane (PM) directly apposed to the chloroplast envelopes (CE) and layers of carotenoid pigment granules (dark gray circles) subtended by thylakoid membrane (TM). The microtubules of the D4 rootlet are arranged in a three-over-one pattern. 
(d) A diagram of a C. reinhardtii cell (as in panel a) illustrating measurements discussed throughout the text and listed in Table I. L, cell length; R1, distance from the basal bodies to the tip of the most extensively acetylated rootlet; R2, distance from the basal bodies to the tip of the second most extensively acetylated rootlet; E1, distance from the basal bodies to the posterior edge of ChR1 photoreceptor–specific fluorescence.

Phototaxis requires specific localization of the eyespot relative to the flagella The eyespot must be located at a specific position relative to the flagellar plane for photoreceptor activation and the resulting influx of Ca2+ to elicit movement in the correct direction. Observation of the eyespot pigment granule layers  revealed that the eyespot was precisely positioned relative to other cytoskeletal structures throughout the cell cycle.   Eyespot placement is dictated by the cytoskeleton, specifically by properties of the D4 rootlet.

1. http://jcb.rupress.org/content/193/4/741

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