Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

miR-7 is activated by EGF-receptor (EGFR) signaling in cells undergoing the initial steps in photoreceptor differentiation. Furthermore, conditional Dicer-null mutation in the developing retina results in apparently normal retinal structure – albeit interspersed with rosettes and fated to progressive degeneration – pointing to the role of miRs in normal photoreceptor genesis and function. Further studies are warranted to fully elucidate the role of miRs in photoreceptors in general and in their development in particular.

Conclusions

When investigating cellular events and molecular mechanisms, the accuracy of models is only as good as the methods used to collect the data and the relevance to human retinal development of the animal model used. Future studies in other vertebrate species (e.g., zebrafish) and in human embryonic stem cells, with more sophisticated tools to dissect cell-fate specification/determination mechanisms, and the analysis of the vast amount of omic data available will permit integration of current photoreceptor development models to more closely represent the relevance to human disease. It is the authors’ auspice that this article be read as the starting point to stimulate the reader’s curiosity to further investigate the field of retinal developmental neurobiology for more in-depth understanding and up-to-date breakthroughs.

See also: Coordinating Division and Differentiation in Retinal Development; Embryology and Early Patterning; Histogenesis: Cell Fate: Signaling Factors; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Retinal Histogenesis; Zebra Fish–Retinal Development and Regeneration.

Further Reading

Adler, R. and Raymond, P. A. (2008). Have we achieved a unified model of photoreceptor cell fate specification in vertebrates? Brain Research 4: 134–150.

Chen, J., Rattner, A., and Nathans, J. (2005). The photoreceptorspecific nulear receptor Nr2e3 represses transcription of multiple cone-specific genes. Journal of Neuroscience 25: 118–129.

Cheng, H., Aleman, T. S., Cideciyan, A. V., et al. (2006). In vivo function of the orphan nuclear receptor NR2E3 in establishing photoreceptor identity during mammalian retinal development. Human Molecular Genetics 15: 2588–2602.

Fishman, R. S. (2008). Evolution and the eye: The Darwin bicentennial and the sesquicentennial of the origin of species. Archives of Ophthalmology 126: 1586–1592.

Hatakeyama, J. and Kageyama, R. (2004). Retinal cell fate determination and bHLH factors. Seminars in Cell and Developmental Biology 15: 83–89.

Hendrickson, A., Bumsted-O’Brien, K., Natoli, R., et al. (2008). Rod photoreceptor differentiation in fetal and infant human retina.

Experimental Eye Research 87: 415–426.

Lamba, D., Nelson, G., Kari, M. O., and Reh, T. A. (2008). Specification, histogenesis, and photoreceptor development in the mouse retina. In: Chalupa, L. M. and Williams, R. W. (eds.) Eye, Retina, and Visual System of the Mouse, pp. 299–310. Cambridge, MA: MIT Press.

Livesey, F. J. and Cepko, C. L. (2001). Vertebrate neural cell-fate determination: Lessons from the retina. Nature Reviews Neuroscience 2: 109–118.

Mears, A. J., Kondo, M., Swain, P. K., et al. (2001). Nrl is required for rod photoreceptor development. Nature Genetics 29: 447–452.

Ng, L., Hurley, J. B., Dierks, B., et al. (2001). A thyroid hormone receptor that is required for the development of green cone photoreceptors.

Nature Genetics 27: 94–98.

Nishida, A., Furukawa, A., Koike, C., et al. (2003). Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nature Neuroscience 6: 1255–1263.

Oh, E. C., Khan, N., Novelli, E., et al. (2007). Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL. Proceedings of the National Academy of Sciences of the United States of America 104: 1679–1684.

Onishi, A., Peng, G.-H., Hsu, C., et al. (2009). Pias3-dependent SUMOylation directs rod photoreceptor development. Neuron 61: 234–246.

Roberts, M. R., Srinivas, M., Forrest, D., et al. (2006). Making the gradient: Thyroid hormone regulates cone opsin expression in the developing mouse retina. Proceedings of the National Academy of Sciences of the United States of America 103: 6218–6223.

Zhang, J., Gray, J., Wu, L., et al. (2004). Rb regulates proliferation and rod photoreceptor development in the mouse retina. Nature Genetics 36: 351–360.

Glossary

Axoneme – The cytoskeletal backbone of cilia and flagella composed of nine microtubule doublets aligned in a cylindrical array. Motile axonemes, generally referred to as 9 + 2 axonemes, contain a pair of central singlets and dynein arms.

Basal body – A centriole that becomes associated with the cell membrane for the nucleation of a cilium. Doublet microtubules of cilia in most animals are extensions of triplets of the basal body.

Centriole – A barrel-shaped or cylindrical organelle composed in most animals of nine triplet microtubules. A pair of centrioles, surrounded by an amorphous zone containing many proteins, constitutes the centrosome.

Cilia – The extensions of the cell surface that have a plasma membrane and contain a cytoskeletal core of microtubules, called an axoneme, which extends out from the basal body. They may be motile, as in the airway epithelium in humans, or nonmotile and sensory in function. Cilia and flagella have the same organization, but flagella are generally motile and are longer.

Intraflagellar transport – A microtubule-based trafficking pathway required for the formation and maintenance of cilia and flagella. Intraflagellar transport (IFT) involves the bidirectional transport of a protein adaptor complex composed of highly conserved IFT proteins. The pathway requires kinesin 2 family motors in the outward (anterograde) direction and a cytoplasmic dynein in the return (retrograde) direction. The pathway is equally important in cilia and flagella. Kinesins – The plus-end-directed motor proteins powered by adenosine triphosphate hydrolysis to walk along microtubules. These are involved in multiple cellular functions, including transport of cargo, mitosis, and meiosis. Many different genes encode kinesins with different properties and functions. Microtubules – The cytoskeleton tubules assembled from protofilaments composed of linear polymers of a- and b-tubulin heterodimers. These are asymmetric hollow cylinders with plus and minus ends that differ in polymerization rate and binding proteins. They form a dynamic network within the cell and organize themselves into complex structures, such centrioles, basal bodies, and cilia.

Microvilli – The membrane protrusion from the cell surface composed of cytoplasm and dense bundles

of actin filaments serving to increase the surface area of the cell.

Sensory cilia – The cilia that contain membrane receptors and signaling components. These often have a nonmotile 9 + 0 axoneme, but actively motile cilia may also serve a sensory function.

Introduction

There are at least two fundamentally different designs for visual photoreceptors, generally referred to as the rhabdomeric type and the ciliary type. Rhabdomeric photoreceptors are widely represented among invertebrates and have been intensively studied in the horseshoe crab, fruit fly, and squid. Rhabdomeric photoreceptors concentrate the visual pigment for photon capture in a highly replicated array of microvilli, each containing an actin cytoskeletal core. The array of microvilli is called the rhabdomere. The visual pigment of ciliary photoreceptors is also concentrated in a photon capture organelle called the outer segment (OS). However, the OS is derived from the plasma membrane of a cilium and retains the microtubule-based cytoskeletal core, called an axoneme, which is common to all cilia. Often, rhabdomeric and ciliary photoreceptors are referred to as invertebrate and vertebrate types, respectively, but this distinction can be misleading. Both photoreceptor types appeared early in animal evolution and are present in multiple invertebrate phyla. Furthermore, some animals have both photoreceptor types, and the retina in the compound eye of the scallop contains a layered juxtaposition of both rhabdomeric and ciliary photoreceptors. Although vertebrate photoreceptors with true rhabdomeres have not been identified, the recent discovery of intrinsically light-sensitive ganglion cells (ipGCs) has led to the realization that their melanopsin photopigment and transduction pathway have features in common with rhabdomeric rather than ciliary photoreceptors. This suggests that ipGCs may have originated from an evolutionary ancient rhabdomeric precursor.

Turnover of the OS and Phototransduction

Machinery

The light-sensitive OS of vertebrate photoreceptors is an elegantly complex organelle that serves as the starting

point for highly sensitive nighttime vision (rods) as well as high acuity, color vision (cones) in the daytime. The OS consists of a stack of membrane disks containing a photopigment (opsin) of the guanine-nucleotide-binding protein-coupled receptor (GPCR) family, and a large array of cytosplasmic, cytoskeletal, membrane, and membraneassociated proteins that are essential for phototransduction. The phototransduction cascade begins with photon absorption by the vitamin-A-derived chromophore of opsin and proceeds through activation of transducin, a guanine-nucleotide-binding protein (G-protein), which in turn activates a phosphodiesterase that reduces cytoplasmic cyclic guanosine monophosphate (cGMP) levels. The photoresponse is a hyperpolarization event that occurs when declining local cGMP levels result in closure of the cGMP-gated channel in the OS plasma membrane. The high sensitivity of the rod cells, which can respond to single-photon absorption events, and the rapid responses of cone cells in bright light, depend on the integrity of the disk stack and close juxtaposition of the protein components of the transduction cascade. Optimal function also depends on other OS proteins that support transduction such as membrane guanylyl cyclases (GC1 and GC2), which regenerate cGMP, a Na+/Ca2+ exchanger which regulates Ca2+ levels, anaerobic glycolysis, which supplies a portion of the adenosine triphosphate (ATP), and the pentose phosphate shunt which supplies nicotinamide adenine dinucleotide phosphate (NADPH), essential for conversion of the all-trans retinaldehyde to retinol in the visual pigment cycle.

Two additional interdependent features of OS organization, essential for optimal visual function, are proper targeting of the phototransduction proteins to the OS and long-term maintenance through OS renewal. A striking feature of normal rods and cones is the high level to which phototransduction proteins are concentrated in the OS. Since those proteins are all synthesized at polyribosomes in the inner segment, a great deal of recent focus has been on those mechanisms that are essential for proper trafficking of OS proteins. This is a major problem throughout the life of the cell because OSs renew at a particularly high rate (see Figure 1). In the 1960s, Richard Young, then at the University of California at Los Angeles, thoroughly documented the fact that rod OSs are renewed through continuous new disk assembly adjacent to the inner segment. Furthermore, Young along with Dean Bok determined that OS length is maintained through a compensatory shedding of disks from the distal tip where they are phagocytized and degraded by the retinal pigment epithelium. In the 1970s, these same concepts were extended to include the cone OSs. The turnover of OSs is highly conserved, but occurs at very different rates in different species. In mice, rod OSs turnover once every 10 days.

The Photosensitive Organelle as a

Sensory Cilium

With the advent of transmission electron microscopy in the 1950s, photoreceptors were an early subject of analysis, particularly by Eduardo de Robertis in Argentina and Kiyoteru Tokuyasu and Eichi Yamada in Japan. Those early studies showed that developing photoreceptors have the basic organization of cilia. During early differentiation, a centriole pair moves to the plasma membrane where one member serves as a template for assembly of a microtubule cytoskeletal structure called an axoneme (Figure 2); the other centriole is generally seen next to the basal body as an accessory centriole. Centrioles consist of an array of nine triplet microtubules and each triplet has an A-, B-, and C-tubule. The A-tubule is a complete microtubule comparable to cytoplasmic microtubules, but the B subtubule is a partial microtubule built on the wall of the A-tubule. Likewise, the C-tubule is built on the wall of B. The centriole that associates with the plasma membrane is called a basal body because it serves as a template for outgrowth of doublet microtubules that form the axoneme. As a consequence of basal body templating, the axoneme grows out as an array of nine doublet microtubules that are direct extensions of the A- and B-tubules of the basal body. The photoreceptor and other sensory cilia are generally said to have a 9 + 0 axoneme, in contrast to motile cilia, which have a 9 + 2 axoneme; the 2 in the latter designation refers to a central pair of single microtubules within the core of the axoneme. Although the 9 + 2 axoneme is found in a wide array of motile cilia, 9 + 0 cilia are sometimes motile. For example, the rotatory cilia of the embryonic Henson’s node have a 9 + 0 axoneme.

As the axoneme elongates in rodents, the plasma membrane expands at the distal end of the cilium, and membrane vesicles and tubules accumulate (Figure 2). At this early stage, the photopigment apoprotein, opsin, is localized within the plasma membrane of the distal end of the cilium, and membrane vesicles and tubules accumulate. This region expands in the early stages of OS formation, quickly taking the form of an orderly stack of membrane disks (Figure 2). Frog OSs, as described by S. E. Nilsson in 1964, exhibit ordered disks from the very beginning of differentiation. At later stages, and, presumably during early development, new membrane disks are thought to form as evaginations of the ciliary membrane. The most proximal part of the cilium emanating from the basal body, called the connecting cilium by Eduardo de Robertis in 1956, connects the photosensitive OS with the cells synthetic machinery in the inner segment; components of the phototransduction machinery in the OS are synthesized in the inner segment and must be transported to the OS through the connecting cilium.

Microtubule

plus end

Disk

Singlet Singlet

Figure 3 Structure of the ciliary axoneme of a mature photoreceptor. Longitudinal view is shown on the right and magnified cross sections at the position of the arrows on the left. The axoneme grows out of the basal body as an array of nine doublet microtubules. The microtubule plus ends extend distally and the minus ends are anchored in the basal body. The region adjacent to the basal body is a transition zone in which doublets are closely linked with the plasma membrane by Y-shaped crosslinkers (lower diagram on left); this region is often referred to as the connecting cilium. The region immediately distal to the transition zone is the site of new disc assembly. Within the OS the doublets loose their B-tubule to become singlets in the distal OS. This is shown as an abrupt transition on the right, but conversion to singlets appears to occur gradually leaving mixtures of singlets and doublets (middle diagram on left). The distal OS only has singlets.

The term connecting cilium refers to that portion of the cilium between the basal body and the disk-forming region of the OS. As originally demonstrated by Pal Ro¨hlich in 1976, the connecting cilium is actually comparable to the transition zone, a structure common to all eukaryotic cilia and flagella (Figure 3). Here, the plasma membrane is closely linked to the doublet microtubules of the axoneme by cross-linkers that extend from the doublet microtubule to the plasma membrane. These cross-linkers are stable structures that remain bound to the axoneme after detergent extraction and link the axoneme to cell surface glycoconjugates through transmembrane connections. Although these microtubule-membrane cross-linkers exhibit a conserved Y-shaped structure across many cilium types, their molecular composition has not been determined. Recently, a number of cilium proteins relevant to human photoreceptor

degenerative disease, such as retinitis pigmentosa guanosine triphosphate (GTP)ase regulator (RPGR) and RPGRinteracting protein 1 (RPGRIP), have been localized to the transition zone and some may be components of the cross-linking structures. Further high-resolution analysis of the composition of cross-linkers may lead to a better understanding of the function of connecting cilium proteins that are relevant to human disease.

Distally, beyond the transition zone, the axoneme extends deep into the OS (Figure 3). This point requires emphasis because the term connecting cilium refers to the link between inner and OS and the term is often used with the implication that this is the entire cilium. However, both early electron microscopic studies and numerous immunocytochemistry studies have shown that photoreceptor axonemes extend through much of the length of the OS (Figure 4); in some cases, they extend all the way to the distal tip. The recent finding of extremely long axonemes in mouse, frog, and zebrafish OSs, along with earlier studies showing much shorter axonemes, suggests that they may vary significantly in their length. The reason for variability in axoneme length observed in various studies is not known. A possible explanation, however, is that the distal axoneme is dynamic and unstable, resulting in some cases in poor preservation for morphological studies. The principles governing these length variations in either rods or cones remain unknown, but are likely to be relevant to the finding that OSs maintain a relatively constant length through many cycles of OS turnover.

Evidence for Intraflagellar Transport in Photoreceptors

Recently our work has demonstrated that the assembly of photoreceptor OSs depends on a highly conserved microtubule-based trafficking pathway called intraflagellar transport (IFT). IFTwas originally discovered in the motile flagella of the green alga Chlamydomonas rheinhartii and quickly extended to the sensory cilia Caenorhabditis elegans. The essential components of IFT are the kinesin and dynein molecular motors that drive movement along axonemal microtubules and multiprotein IFT particles that are thought to link cargo such as cytoskeletal and phototransduction proteins to the IFTmotors (see Figure 5). For example, at least 16 different proteins assemble to form two large protein complexes referred to as IFT particles (Figure 5), and all of these proteins are highly conserved between C. rheinhartii and man. IFT transport is bidirectional. In anterograde IFT plus-end-directed motors of the kinesin 2 family move IFT particles with attached cargo toward the plus end of the axoneme, while in retrograde IFT a minus-end-directed dynein motor returns the IFT machinery for exchange with a pool in the cell body. Again, the pathway is highly conserved in that the same molecular

motors identified in C. rheinhartii and C. elegans perform similar functions in virtually all eukaryotic cilia including those in man. For example, heterotrimeric kinesin II, assembled from the kinesin family member 3A and 3B (KIF3A, KIF3B) along with kinesin-associated protein 3 (KAP3) proteins, is the canonical anterograde motor, while a dynein containing the cytoplasmic dynein 2 heavy chain (DNCH2) is the canonical retrograde motor.

A prominent role for IFT in photoreceptor OS formation is now well established. Four of the IFT proteins (IFT88, IFT57, IFT52, and IFT20) have been localized along photoreceptor axonemes and photoreceptor OS assembly defects have been fully characterized in rods of mice with a mutation in IFT88. This has been extended to cone cells with targeted, cone-specific deletion of IFT20, which results in disrupted OS assembly. A central feature of the IFTmodel is the multi-protein IFT particle (Figure 5). IFT particles containing IFT88, IFT57, IFT52, and IFT20 can be isolated from bovine photoreceptor outer segments. The photoreceptor IFT particle is large, fractionating in sucrose gradients at a peak size of 500–750 kDa and has properties remarkably similar to those originally described in C. rheinhartii. This implies that the photoreceptor particle contains additional IFT proteins for which antibodies have not yet been generated. Evidence for photoreceptor IFT is also based on analysis of its canonical motors. All three subunits of kinesin II as well as DNCH2 have been localized to photoreceptor axonemes. Furthermore, conditional deletion of the KIF3A subunit of kinesin II disrupts OS assembly, causes rhodopsin mislocalization, and results in photoreceptor cell death.

A Special Role for KIF17 in

Photoreceptors

The foregoing description of photoreceptor IFT describes conditions and expectation of a canonical IFTmodel that has

applicability in virtually all ciliated cells. However, a novel feature of photoreceptor IFT is the critical involvement of an additional kinesin motor, the homodimeric kinesin family member 17 (KIF17). As illustrated in Figure 5, both kinesin II and KIF17 are associated with the IFT particle along doublet microtubules in the proximal OS, but KIF17 alone is associated with movement of the IFT particle along singlet microtubules in the distal OS. Our recent work has demonstrated that KIF17 is required to form outer segments, but does not simply replace kinesin II function; both kinesins are required. An interesting feature of this work is that while reduced kinesin II function disrupts both photoreceptor and kidney cilium elongation, knockdown of KIF17 results in failed OS assembly with no apparent effect in the kidney. The importance of KIF17 is likely related to the presence of singlet microtubule extensions in photoreceptors (see Figure 3). While singlet extensions are prominent in photoreceptor cilia, as originally illustrated in the older EM literature, singlet extensions in kidney sensory cilia are either very short or not present at all.

Work from the laboratory of Jonathan Scholey at the University of California at Davis has shown that the C. elegans KIF17 homolog, osmotic avoidance abnormal protein (OSM-3), is also required for sensory cilium elongation, specifically in cilia with singlet extensions. In C. elegans cilia OSM-3 serves as an accessory IFT motor along with kinesin II. Specifically, either kinesin motor can function and compensate for loss of the other motor in the proximal cilium, which contains doublet microtubules, but only OSM-3 can extend and move along the distal singlets. In fact, it was the existence of singlet extensions in photoreceptors that drew our attention to KIF17 and led to our finding of a prominent role of this accessory kinesin motor. However, the simple model for dual IFT kinesins in C. elegans does not fully explain findings in photoreceptors. The C. elegans model predicts that knockdown of KIF17 would result in short OSs that fail to elongate.

Anterograde Retrograde

Dynein

KIF17

Kinesin II

IFT particle

Cargo

Figure 5 Conceptual diagram of intraflagellar transport (IFT) in photoreceptors. (Left) Anterograde IFT uses the plus end directed kinesin motors, kinesin II and KIF17, to move IFT particles with attached ’cargo’ toward the distal, plus end of the axoneme (arrow). Note that the minus-end-directed motor, dynein, is illustrated as cargo for anterograde IFT. Based largely on work in C. elegans, it is hypothesized that kinesin II and KIF17, move cooperatively on doublets in the proximal outer segment and that KIF17 operates alone on singlets in the distal outer segment. (Right) Retrograde IFT uses a cytoplasmic dynein motor to return IFT particles and cargo to the inner segment. Note that kinesin II and KIF17 are illustrated as cargo for retrograde IFT. IFT complexes along with cargo and molecular motors are thought to assemble into large complexes in the inner segment in the region adjacent to the basal body.

Results from direct comparison of mutated forms of the KIF3B subunit of kinesin II and KIF17 suggest that the motors carry out nonredundant functions and suggest that, in addition to a role for KIF17 in distal cilium extension, the motor may also be involved in transport of proteins that are essential for disk assembly in the proximal outer segment. Although knockdown of KIF17 results in ablation of OS formation, a recent mutagenesis study of a

consensus calcium, calmodulin kinase II (CaMKII) phosphorylation site in the KIF17 tail region, demonstrates that KIF17 plays a critical role in the distal OS in the control disk shedding (see Figure 1).

What Is the IFT Cargo?

The requirement for IFT in ciliogenesis has led to the general idea that the IFT particle serves as an adaptor to link IFT cargo to the requisite IFT motors. Some of the early evidence for cargo relates to the motors themselves. Since the axoneme plus end is at the distal end of the cilium, the minus-end-directed dynein motor required for retrograde IFT cannot move from the cell body to the distal tip on its own and available data suggest that it is cargo during anterograde IFT. Likewise, recycling of the plus-end-directed kinesins back to the inner segment appears to require retrograde IFT (see Figure 5). Another feature of axoneme organization is that its microtubules can assemble or disassemble only at the plus end, which is located distally. Since the axoneme can both elongate and shorten, its building blocks are likely cargo in both directions. It is important to point out that since the axoneme provides the principle cytoskeletal scaffold for the OS, IFT would be of great significance if its main purpose were to maintain axoneme structure. Recent evidence, however, suggests that IFT transports membrane components of the OS as well.

The photoreceptor OS provides a special challenge in that the components of phototransduction are present in high abundance and turn over rapidly. The question is: Which of these components is moved to the OS by IFT? One reasonably clear conclusion is that not all OS components require IFT. For example, some phototransduction proteins, such as arrestin and transducin, move between inner and OS in relationship to light and dark adaptation and a strong case can be made for free diffusion as the underlying mechanism for movement of abundant freely soluble proteins. Current evidence favors the idea that many membrane and membrane-associated proteins may require IFT. In C. rheinhartii and C. elegans, where real time imaging is feasible, movement of specific membrane proteins has been detected along the cilium. Rigorous proof that a particular protein is IFT cargo requires direct demonstration that it is linked to the IFT machinery and moves with IFT components, and there are no examples of this type of evidence outside of

C. rheinhartii and C. elegans.

Nonetheless, mutation of the KIF3A subunit of the kinesin II motor, and mutation of the IFT88 subunit of the IFT particle both result in rhodopsin mislocalization, suggesting that rhodopsin is carried as IFT cargo. In support of the specificity of this effect, we recently demonstrated that expression of a dominant-negative

form of the KIF3B subunit of kinesin II in zebrafish cones results in cone opsin mislocalization, but dominantnegative KIF17 blocks OS elongation without causing opsin mislocalization. Since studies of protein mislocalization in cells carrying mutations is an inherently indirect measure, we have recently used a variety of pull-down assays to isolate IFT protein complexes containing the two kinesin motors as well as rhodopsin and another OS membrane protein, retinal guanylyl cyclase 1 (RetGC1 also called GUCY2E). The work with GC1 is particularly interesting because we have identified a small chaperone protein (DNAJB6) that binds specifically to IFT88 in the IFT particle and to the kinase homology domain in the cytoplasmic tail region of GC1. We have proposed that this is the key linkage required for association of membrane GC1 with the IFT machinery and that the binding can be regulated directly through the ATPase activity of heat-shock cognate protein 70 (HSC70). This represents the first specific model for the molecular linkage of a cargo protein with the IFT machinery.

Summary and Perspective

The fact that photoreceptor OSs are sensory cilia has been known for many years, but recent advances in understanding the mechanisms underlying ciliogenesis, including the IFT pathway, are likely to have a large impact on our understanding of the cell biology of photoreceptors. Although mutations in genes involved in ciliogenesis often cause embryonic lethal phenotypes and will not frequently appear among the causes of photoreceptorspecific degenerative disease, our understanding of disease mechanisms are likely to improve through understanding which photoreceptor proteins are IFT cargo. For example, human GC1 contains three human disease causing mutations in the domain that we have recently shown to bind a linker chaperone that couples GC1 to the IFT machinery. This has led us to propose that those human mutations lead to abnormal ciliary trafficking. The importance of photoreceptor cilia also provides a basis for understanding syndromic diseases such a Bardet–Biedl syndrome, which includes RP among a complement of other abnormalities. Such diseases are now referred to as ciliopathies because cilia provide a common basis for understanding pathology in the different tissues that are affected. Finally, placement of photoreceptor OSs in their appropriate niche as a special type of sensory cilium provides evolutionary perspective on pathways common to all cilia that emerged early in eukaryote evolution.

See also: Circadian Photoreception; Genetic Dissection of Invertebrate Phototransduction; Limulus Eyes and Their Circadian Regulation; Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors; Microvillar and Ciliary Photoreceptors in Molluskan Eyes;

The Photoresponse in Squid; Phototransduction: Adaptation in Rods; Phototransduction in Limulus Photoreceptors; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Phototransduction: The Visual Cycle; Retinal Degeneration through the Eye of the Fly; Rod and Cone Photoreceptor Cells: Outer Segment Membrane Renewal.

Further Reading

Baker, S. A., Freeman, K., Luby-Phelps, K., Pazour, G. J., and Besharse, J. C. (2003). IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia. Journal of Biological Chemistry 278: 34211–34218.

Besharse, J. C. and Horst, C. J. (1990). The photoreceptor connecting cilium. A model for the transition zone. In Bloodgood, R. A. (ed.)

Ciliary and Flagellar Membranes, pp. 389–417. New York: Plenum. Bhowmick, R., Li, M., Sun, J., et al. (2009). Photoreceptor IFT

complexes containing chaperones, guanylyl cyclase 1, and rhodopsin. Traffic 10(6): 648–663.

De Robertis, E. (1960). Some observations on the ultrastructure and morphogenesis of photoreceptors. Journal of General Physiology 43(6): supplement, 1–13.

Eckmiller, M. S. (1996). Renewal of the ciliary axoneme in cone outer segments of the retina of Xenopus laevis. Cell Tissue Research

285: 165–169.

Hong, D. H., Yue, G. H., Adamian, M., and Li, T. S. (2001). Retinitis pigmentosa GTPase regulator (RPGR)-interacting protein is stably associated with the photoreceptor ciliary axoneme and anchors RPGR to the connecting cilium. Journal of Biological Chemistry

276: 12091–12099.

Inglis, P. N., Ou, G., Leroux, M. R., and Scholey, J. M. (2007). The sensory cilia of Caenorhabditis elegans. WormBook March 8: 1–22.

Insinna, C. and Besharse, J. C. (2008). Intraflagellar transport and the sensory outer segment of vertebrate photoreceptors. Developmental Dynamics 237: 1982–1992.

Insinna, C., Pathak, N., Perkins, B., Drummond, I., and Besharse, J. C. (2008). The homodimeric kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment development. Developmental Biology 316: 160–170.

Maerker, T., van Wijk, E., Overlack, N., et al. (2008). A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells. Human Molecular Genetics 17: 71–86.

Marszalek, J. R., Liu, X., Roberts, E. A., et al. (2000). Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors. Cell 102: 175–187.

Pazour, G. J., Baker, S. A., Deane, J. A., et al. (2002). The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance. Journal of Cell Biology 157: 103–113.

Rohlich, P. (1975). The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia. Cell Tissue Research 161: 421–430.

Tokuyasu, K. and Yamada, E. (1959). The fine structure of the retina studied with the electron microscope. IV. Morphogenesis of outer segments of retinal rods. Journal of Biophysical and Biochemical Cytology 6: 225–230.

Young, R. W. (1967). The renewal of photoreceptor cell outer segments.

Journal of Cell Biology 33: 61–72.

Young, R. W. and Bok, D. (1969). Participation of the retinal pigment epithelium in rod outer segment renewal process. Journal of Cell Biology 42: 392–403.

Glossary

Arhabdomeral lobe – The proximal segment of the photoreceptor cell containing the nucleus and other organelles.

Arrestin – A protein that binds to metarhodopsin and arrests phototransduction by inhibiting metarhodopsin activation of its target gaunine nucleotide-binding protein.

Calpain-like protease – An enzyme isolated from squid photoreceptors that, like calpain, requires millimolar concentrations of calcium for its proteolytic activity.

Metaretinochrome – The conformation of retinochrome when retinal has been photoisomerized to 11-cis-retinal.

Metarhodopsin – The light-activated conformation of opsin when bound to all-trans retinal. Retinochrome – A photosensitive protein that binds all-trans retinal.

Rhabdomeral lobe – The distal segment of the photoreceptor cell containing the molecular machinery of phototransduction.

Rhabdomere – The collective surfaces of the distal segment composed of densely packed microvilli. Rhodopsin kinase – An enzyme that adds phosphates onto serine or threonine residues in the carboxyl-terminus of metarhodopsin.

Introduction

Squid, like most invertebrates, have light-sensing organs. The visual systems of squid are composed of camera-type eyes. Incoming light passes through a single lens and an image is formed on the light-sensing cells of the retina in the anterior chamber of the eye. The retina consists of a single layer of photoreceptive neurons that are segmented in structure. The outer segment, also known as the rhabdomeral lobe, contains the protein machinery of phototransduction. The inner segment, or the arhabdomeral lobe, contains the cellular organelles involved in protein synthesis and the soma contains the cell nucleus. Axons arising from the soma of the photoreceptors comprise the optic nerve that projects to the squid brain (Figure 1).

The photoreceptor outer-segment membrane forms densely packed microvilli that greatly increase the surface area of the membrane available to capture incoming photons of light (see enlargement in Figure 1). Embedded in the membrane are the rhodopsin receptors containing light-sensitive chromophores. Light activation of rhodopsin sets off a cascade of molecular interactions that culminate in depolarization of the photoreceptor membrane (Figure 2). In recent years, many of the molecular components of the light-activated signaling system have been identified and a review of our current knowledge of these components is outlined here. Equally important to vision are the molecular mechanisms that inhibit signal transduction after transmission of the light signal (Figure 3). These mechanisms are not well understood; however, many of the components of the inactivation pathway have been revealed and these will also be discussed.

Molecular Components of Squid Visual

Signal Transduction

The squid visual signal transduction system is composed of a light-sensitive receptor, rhodopsin, that transduces its activation signal via a heterotrimeric G protein (Gq) to a phospholipase C (PLC) enzyme. Activated phospholipases hydrolyze membrane phospholipids liberating soluble inositiol 1,4,5-trisphosphate (IP3) and membranebound diacylglycerol (DAG). While it is still not clear how these second messengers stimulate membrane depolarization, it probably involves release of calcium from the submicrovillar tubules as a result of IP3 stimulation of receptors on these organelles and perhaps direct stimulation of transient receptor potential (TRP)-like channels in the membrane by DAG. Together, these mechanisms raise intracellular calcium and increase membrane depolarization.

Squid Rhodopsin

Squid rhodopsin consists of a guanine nucleotide-binding protein-coupled receptor (GPCR) or opsin bound to a light-absorbing retinoid chromophore. Squid opsin genes have been cloned from Loligo forbesi, Loligo pealei, and

Todarodes pacificus, and they share 45% sequence identity with other invertebrate and vertebrate opsins. However,

squid opsins are about 100 residues larger than those of vertebrates, primarily owing to the addition of a prolinerich carboxyl-terminal tail. Squid rhodopsins bear structural hallmarks of the GPCR superfamily, including

amino-terminal sites of N-linked glycosylation, carboxylterminal sites of palmitoylation, a disulfide bridge between two extracellular loops, proline residues in the a-helices of the transmembrane domains, and a (D/E)R(Y/W)