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

often oriented near the horizontal plane, and quite variable depending on the background (Figure 3). Nevertheless, the background polarization can be used to enhance the contrast of the visual world and make midwater objects more visible. Squids take advantage of this, making them more effective predators on fishes and transparent planktonic prey against a polarized background than against a depolarized one. Presumably, they use their polarizedlight sense to detect differences in polarization between the prey and the background, making objects of similar overall brightness appear distinguishable.

If squids and cuttlefishes can distinguish objects based on polarization, it should not be surprising that they also have body patterns that reflect polarized light, undetectable by many marine animals, and apparently employ these patterns to communicate with each other. The polarization patterns are actively controlled by the animal producing them and can appear and disappear in fractions of seconds. When displayed, the polarization reflections remain highly visible to a conspecific individual even as the signaler changes its posture or moves its arms about.

Two other groups of animals, one marine and one terrestrial, are currently known to recognize and respond to polarization signals. Mantis shrimps produce an abundance of signals based on patterns of polarized light reflected from their carapace (Figure 6), and use these signals during mating and aggressive displays. Like the cephalopods, of course, they are marine invertebrates and conduct their diplays underwater. One group of insects, however, uses polarized-light signals in the open air. Many species of tropical butterflies find mates in the diffuse light under the rainforest canopy. Here, the background polarization is relatively weak, and the strong polarization pattern of the sky is rarely visible. Thus, the polarization produced by reflection from scales on butterfly wings can act as an unusually strong, visible signal.

Sensitivity to Circularly Polarized Light

This discussion of polarized-light sensitivity would not be complete without mention of a recently discovered visual modality, sensitivity to circularly polarized light. Circular polarization differs from linear polarization, the type discussed exclusively until now, in that the e-vector does not remain within a single plane, but instead rotates around the axis of the beam of light. Circularly polarized light is not common in nature, and its presence cannot be detected with standard polarization-sensing systems. Despite this, one group of animals, the mantis shrimps, perceives circularly polarized light and produces circularly polarized signals by reflection. This ability is particularly unexpected because there is no known source of circular polarization underwater other than signals from other mantis shrimps, so it is difficult to explain how and why the ability originally arose. It is possible that circular polarization sensitivity in these animals first appeared as an accidental epiphenomenon related to the unusual way in which their linear polarization system is assembled, and that this led to the elaboration of signals based on circularly polarized light. See the suggested reading for a more detailed account of this unusual finding.

Summary

The ability to perceive and respond to linearly polarized light is widespread among animals, occurring in many vertebrates and invertebrates. Some of these species use polarization for general tasks that do not require precise imaging, such as finding water or navigating using patterns of scattered polarized light in the sky. Others truly see polarized objects and use this imaging ability to detect prey and recognize signals from conspecifics. Our poor understanding of the biology of polarized-light sensitivity

in vertebrates, and the recent discovery of circular polarization sensitivity in mantis shrimps, suggest that there are other aspects to polarized-light sensitivity and to polarization vision that still remain to be revealed.

See also: Microvillar and Ciliary Photoreceptors in Molluskan Eyes; The Photoresponse in Squid; Phototransduction: Rhodopsin; Rod and Cone Photoreceptor Cells: Inner and Outer Segments; The Colorful Visual World of Butterflies; Evolution of Opsins.

Further Reading

Chiou, T. -H., Kleinlogel, S., Cronin, T. W., et al. (2008). Circular polarisation vision in a stomatopod crustacean. Current Biology 18: 429–434.

Cronin, T. W., Shashar, N., Caldwell, R. L., et al. (2003). Polarization vision and its role in biological signaling. Integrative and Comparative Biology 43: 549–558.

Dacke, M., Doan, T. A., and O’Carroll, D. C. (2001). Polarized light detection in spiders. Journal of Experimental Biology 204: 2481–2490.

Hawryshyn, C. W. (1992). Polarization vision in fish. American Scientist 80: 164–175.

Horva´th, G. and Varju´, D. (2004). Polarized Light in Animal Vision: Polarization Patterns in Nature. Berlin: Springer.

Muheim, R., Phillips, J. B., and Akesson, S. (2006). Polarized light cues underlie compass calibration in migratory songbirds. Science 313: 837–839.

Shashar, N., Rutledge, P., and Cronin, T. W. (1996). Polarization vision in cuttlefish: A concealed communication channel? Journal of Experimental Biology 199: 2077–2084.

Sweeney, A., Jiggins, C., and Johnsen, S. (2003). Polarized light as a butterfly mating signal. Nature 423: 31–32.

Waterman, T. H. (1981). Polarization sensitivity. In: Autrum, H. (ed.)

Handbook of Sensory Physiology VII/6B, pp. 281–469. Berlin: Springer.

Wehner, R. (2001). Polarization vision – a uniform sensory capacity?

Journal of Experimental Biology 204: 2589–2596.

Glossary

Cell polarity – The asymmetry in cell shape, protein distributions, and cell functions.

Golgi complex – A biosynthetic organelle comprised of a stack of membranous cisternae that are involved in protein modifications, such as processing of N-linked sugars as well as sorting and transport of membrane proteins. Newly synthesized membrane and secretory proteins enter the stack through the cis-Golgi, progress through the medial cisternae and exit at the trans-Golgi.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) – An essential second messenger as well as lipid regulator of membrane trafficking. Along with its precursor phosphatidylinositol-4-phosphate (PI(4)P), it regulates Arfs and is regulated by them, providing a positive-feedback loop that regulates membrane trafficking.

Post-Golgi transport carriers (TCs) – The postGolgi vesicles that carry cargo to the plasma membrane. It is now clear that these carriers are large pleiomorphic structures, rather than small vesicles, as previously believed, thus the term vesicles has been replaced with transport carriers. Small GTPases – The members of the low- molecular-weight (20–25 kDa) Ras super family of guanosine-triphosphate (GTP)-binding proteins comprised of at least four large families, including the Arfs and the Rabs. Small GTPases function by providing directionality to membrane traffic through the molecular switch whose ON and OFF states are triggered by binding and hydrolysis of GTP. The nucleotide-bound state determines the affinity of interactions with regulatory proteins and the downstream effectors of small GTPases.

SNARE proteins – The soluble N-ethylmaleimide- sensitive factor attachment protein receptor (SNARE) proteins are major components of the intracellular machinery responsible for targeted membrane delivery. SNAREs were identified

as membrane receptors for the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) in the cell-free system that reconstituted intra-Golgi trafficking. SNARE proteins form complexes, which are generally composed of a four helical bundle that bridges opposing membranes and brings them into close proximity to initiate fusion.

The trans-Golgi network (TGN) – A tubular network in the close proximity to the trans-Golgi cisternae that represents the central sorting station of the cell, where proteins and lipids destined for different subcellular domains are segregated from each other and sorted into post-Golgi TCs.

Introduction

Retinal rod photoreceptors are exquisitely complex polarized cells that carry photon detection and visual transduction that are essential as the first step in vision. Following the final cell division of their precursors, photoreceptors attain a level of polarity that is nearly unmatched in other cells of the body. Maintaining this organization throughout the lifetime of the organism is a prerequisite for vision. Proper targeting and retention of the macromolecular complexes involved in the visual transduction cascade are accomplished by the highly coordinated action of protein and lipid regulators that together constitute the membrane trafficking machinery. Specific components of this machinery involved in the directed delivery of rhodopsin and its associated proteins and lipids are only beginning to emerge.

Photoreceptor Polarity

Rod photoreceptors are modified neurons with specialized light-sensing organelle, the rod outer segment (ROS). The ROS is filled with membranous disks housing the phototransduction machinery that converts photon absorption by rhodopsin into changes in neurotransmitter release, thus transmitting photosensory information to the visual cortex. The light-sensing machinery is comprised of peripheral and integral membrane proteins of the ROS. It is continuously replenished through ROS disk membrane renewal, followed by its removal through daily shedding and phagocytosis by retinal pigment epithelial (RPE) cells.

The ROS disk membrane proteins are embedded in a low-viscosity lipid bilayer milieu comprised of unsaturated long-chain phospholipids highly enriched in omega-3 docosahexaenoic acid (DHA, 22:6(n-3)), which is essential for sensory membrane function and for cell

survival. The exceptionally high content of polyunsaturated DHA phospholipids renders ROS membranes highly susceptible to light and oxidative damage.

ROSs initially form from primary cilia, and a short 9+0 (nonmotile) connecting cilium remains in the adult as the only path of communication between the ROS and the photoreceptor rod inner segment (RIS). The RIS houses mitochondria and biosynthetic membranes involved in oxidative metabolism and membrane protein and lipid biosynthesis, respectively. The photoreceptor-connecting cilium corresponds to the transition zone of primary cilia, which is considered a gateway for the admission of specific proteins to this privileged intracellular compartment.

The primary cilium is the site of assembly of large molecular complexes involved in intraflagellar transport (IFT). IFT protein 20 (IFT20) subunit links mammalian IFT complex with the microtubule motor, kinesin II. The base of the cilium is a region of particularly high lipid ordering, separating the ciliary membrane from the surrounding plasma membrane due to high cholesterol content and glycosphingolipid products of the phosphatidylinositol 4-phosphate (PI(4)P)- and Arf-dependent effector four-phosphate-adaptor protein 2 (FAPP2). Cholesterol rings have also been reported to surround the photoreceptor connecting cilium. Lipid ordering might be important for the docking of the basal body to the plasma membrane or the extension of the ciliary axoneme, both essential processes in ciliogenesis.

The RIS is separated from the nuclear and synaptic domains by adherens junctions (AJs) that comprise a continuous adhesion belt, the outer limiting membrane (OLM). Interestingly, this junctional region lacks tight junctions that normally confine plasma membrane proteins to their respective domains. Nonetheless, rod membrane proteins are strictly confined to their specialized domains and the maintenance of polarity is essential for the cell’s function and survival. The photoreceptor synaptic terminal contains specialized ribbon synapses that are responsible for the tonic release of neurotransmitters, which is interrupted by photon capture.

Photoreceptor cytoskeletal networks and molecular motors play a major role in the cell polarity. Microfilaments provide structural support by encircling the RIS beneath the plasma membrane and are anchored at the AJs. Other polar dynamic actin networks and filaments are also dispersed through the cell, most notably in the distal portion of the cilium, at the sites of ROS disk formation where they regulate the growth of nascent disks. Actinbased motility through the cilium is thought to be mediated by myosin VIIa, the product of the Usher syndrome (USH) 1B (Usher1B) gene. An array of microtubules radiates into the RIS from the microtubule-organizing center nucleated by a pair of centrioles located below the cilium. Microtubules generate polar networks that generally determine the position of membrane organelles

and allow intracellular motility. Heterotrimeric molecular plus end-directed motor kinesin-II and homodimeric KIF17 mediate microtubule-dependent trafficking into the ROS. The absence of KIF3A subunit of kinesin-II causes membrane accumulation in the RIS and cell death. Cytoplasmic dyneins 1 and 2 mediate retrograde trafficking to the centrosome from the RIS and the ROS, respectively.

Photoreceptor Biosynthetic Membrane

Trafficking: Endoplasmic Reticulum,

Golgi, and Post-Golgi Transport Carriers

ROS integral membrane proteins are synthesized on the rough endoplasmic reticulum (ER), pass through the Golgi apparatus, and are then incorporated into post-Golgi vesicles or, in current terminology, rhodopsin transport carriers (RTCs) that bud from the trans-Golgi network (TGN), the central membrane sorting station of the cell, and are transported to a docking site near the base of the cilium. Along with rhodopsin, DHA phospholipids are co-transported on RTCs, which fuse with the specialized domain separating the ciliary membrane from the surrounding RIS plasma membrane, thereby regulating the replenishment of light-sensitive ROS membranes. Rhodopsin represents 90% of the newly synthesized protein in rod photoreceptors, and its biosynthetic pathway is best understood. In the 1980s, the laboratories of David S. Papermaster and Joseph C. Besharse combined electron microscope (EM) immunocytochemistry, autoradiography, and freeze-fracture analysis and demonstrated that newly synthesized rhodopsin is transported vectorially to the base of the cilium on membranous carriers. Membrane biosynthesis was also studied by pulse–chase experiments that established the kinetics of movement of newly synthesized rhodopsin through membrane compartments separated by subcellular fractionation. This methodology was subsequently refined by Deretic and Papermaster to incorporate high-resolution linear sucrose gradients, which separated the low-density post-Golgi carriers from other subcellular organelles, including the Golgi, TGN, plasma membrane, and synaptic vesicles.

Successful isolation of post-Golgi RTCs provided not only insight into their molecular composition, but was also the basis for the development of the retinal cell-free assay that reconstitutes RTC budding in vitro. The development of this assay in our laboratory led to the discovery that rhodopsin contains a sorting signal within its five C-terminal amino acids that regulates its incorporation into RTCs as they bud from the TGN. Abundant evidence points to the role of the amino acid sequence valine-x- proline-x (VxPx motif) in rhodopsin’s C-terminal domain as a sorting signal into RTCs and delivery to the cilium and the ROS. In our retinal cell-free system a monoclonal antibody, whose antigenic site is within the five C-terminal

amino acids of rhodopsin, and synthetic peptides corresponding to the C-terminus of rhodopsin inhibit RTC budding from the TGN. Studies in a number of laboratories employing transgenic animals expressing rhodopsin lacking the sorting signal, or rhodopsin–C-terminal fusion proteins carrying autosomal dominant retinitis pigmentosa (adRP) mutations, confirmed that the absence of the correct sorting information results in the targeting to the RIS plasma membrane and the synapse in vivo.

Because of the exceptionally high membrane turnover, photoreceptors are vulnerable to mutations that affect membrane trafficking. Among the rhodopsin mutations with the most severe phenotypes are those that alter the rhodopsin C-terminal VxPx targeting motif. Rhodopsin C-terminal mutations cause rapid photoreceptor cell death, retinal degeneration, and blindness in adRP. Within the VxPx targeting motif, V345 and P347 are the primary sites of C-terminal adRP mutations involving single amino acid substitutions.

To dissect the sorting machinery that regulates RTC budding, targeting, and fusion, it was essential to identify the resident proteins of this organelle. These studies indicated a succession of binding on RTCs of members of the small guanosine triphosphate (GTP)-binding protein families (G proteins or small GTPases) that are the known regulators of membrane trafficking.

Small GTPases of the Rab and Arf Families

and Their Regulators in Rhodopsin

Trafficking

Rabs

Directed delivery of membrane cargo is mediated through vesicular transport regulated by the small GTPases of the Rab and Arf families, which play a central role in organizing intracellular membrane trafficking. All GTPases function by providing a universal molecular switch whose ON and OFF states are triggered by binding and hydrolysis of GTP. Small GTPases are in a dynamic equilibrium between the cytosol and the membranes maintained by the interactions with a number of regulatory proteins, including nucleotide exchange factors (GEFs), GTPaseactivating proteins (GAPs), and their downstream effectors, and the affinity of these interactions is determined by the nucleotide-bound state. The Rab family of small GTPases includes over 60 members that are generally designated numerically (i.e., Rab6, Rab8, and Rab11). Upon GTP binding, activated Rabs recruit a multitude of effectors that organize membrane domains involved in the tethering of membranes to other membranes and to cytoskeletal elements, thus conferring directionality to membrane traffic. Macromolecular complexes organized by Rabs provide a unique identity to membrane microdomains within cellular organelles. Consequently, a change in Rabs,

termed Rab conversion, changes the membrane identity and accompanies cargo progression through intracellular compartments.

Rab6 regulates retrograde transport between the Golgi and the ER, but in cells with hypertrophied synthesis of simple membranes it also associates with post-Golgi carriers. In photoreceptor cells, Rab6 is associated with the Golgi, TGN, and RTCs. It was demonstrated in Drosophila that Rab6 regulates rhodopsin trafficking, and that the expression of the GTPase-deficient mutant of Rab6 leads to retinal degeneration.

Rab11 has been localized to both the Golgi and the recycling endosomes, which are involved in return of plasma membrane receptors to the cell surface through endocytosis. However, the Golgi-associated function of Rab11 is less well understood. The specific functions of Rab11 in apparently divergent cellular processes are probably based on its ability to interact with different effector molecules that belong to the family of Rab11interacting proteins (FIPs), which are localized in different trafficking pathways. Rab11 is associated with photoreceptor TGN and RTCs where it interacts with FIP3. At the TGN, Rab11 and FIP3 are incorporated into a ciliary targeting complex regulated by the small GTPase Arf4, described below. Sustained presence of Rab11 on RTCs suggests that Rab11 might also interact with the conserved octameric Sec6/8 complex, also known as the exocyst in yeast. The Sec6/8 complex tethers the Rab11/FIP3positive membranes and is involved in tethering RTCs to the RIS plasma membrane. Exocyst complex localizes to the cilia in polarized epithelial cells, and we also find it at the base of the photoreceptor cilium. In Drosophila, interaction of the Sec6/8 complex with Rab11 plays a role in the tethering of membranes carrying rhodopsin. Rab11 may also cooperate with another RTC-associated Rab, Rab8. A handover from Rab11 to Rab8, or Rab conversion, may occur at the base of the cilium to couple the final stages of traffic along the ciliary pathway.

Rab8 regulates polarized trafficking in epithelial cells and neurons through its activity on cytoskeleton remodeling necessary for membrane outgrowth and the formation of cellular protrusions. It has recently emerged as a major player in ciliogenesis. In retinal photoreceptors, Rab8 regulates RTCs fusion and ROS biogenesis. It acts at the base of the cilium in conjunction with another small GTPase (Rac1), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), actin and the phosphoinositideand actinbinding protein moesin. In addition, Sec6/8 complex is also likely to function as one of Rab8 effectors in ciliogenesis. Transgenic Xenopus with photoreceptor-specific expression of GFP-Rab8Q67L dominant-active mutants show normal photoreceptor cell morphology, but cause slow retinal degeneration, whereas GFP-Rab8T22N GTPase-deficient dominant-negative mutants show a defect in membrane tethering and accumulate RTCs in

the vicinity of the cilium, leading to rod cell death and rapid retinal degeneration. Emerging evidence points to Rab8 as a central regulator of the biogenesis of primary cilia, suggesting that its regulation of rhodopsin trafficking may be a part of a broad and more general role for Rab8 in the regulation of ciliogenesis.

Mutations that affect regulatory proteins of small GTPases and their interacting proteins have been found to cause X-linked retinitis pigmentosa (RP), autosomal recessive Leber congenital amaurosis (LCA), and choroideremia. Rab escort protein 1 (Rep1), encoded by the choroideremia gene is a subunit of geranylgeranyl transferase, the enzyme that isoprenylates Rab proteins. Rab8interacting proteins, Optineurin and Huntingtin, are also linked to the neurodegenerative diseases primary openangle glaucoma (POAG) and Huntington’s disease, respectively. Rab8, Optineurin, and Huntingtin are collectively involved in the linkage of membrane organelles to the cytoskeleton, suggesting that the breakdown of this linkage may be a common theme in retinal degeneration and in other neurodegenerative diseases.

Arfs

The Arf family of small GTPases includes three different groups of proteins: the Arfs, Arf-like proteins (Arls), and SARs. Arfs were originally discovered as ADPribosylation factors, but in 2006 the new nomenclature for the human Arf family of GTP-binding proteins, formerly known as ARF, ARL, and SAR proteins, has been established. Arfs are no longer called ADP-ribosylation factors (ARFs), since ADP-ribosylation appears unrelated to their physiological function. Arf family members regulate membrane trafficking, lipid metabolism, organelle morphology, and cytoskeleton dynamics. These functions were elucidated for the abundant class I Golgi Arfs, Arf1 and Arf3, and the plasma membrane-associated Arf6. The loss of a single class I or class II Arf has little effect on membrane trafficking, but the deletion of pairs of Arfs causes distinct defects. This suggests a pair-wise engagement of Arfs and a certain redundancy in their function. Arf function depends on GTP hydrolysis mediated by Arf GAPs, which are essential for coupling the proofreading of cargo incorporation to the budding of membrane carriers and are often incorporated into protein coats.

The selection and packaging of sensory receptors and membrane cargo targeted to the primary cilia and ciliaderived sensory organelles are critical to replenish the ciliary membrane, yet it remains poorly understood. Our recent studies have demonstrated that rhodopsin C-terminal VxPx targeting signal binds Arf4 to regulate incorporation of rhodopsin into RTCs at the TGN. The function of the class II Golgi-associated Arfs, Arf4 and Arf5, is least understood, yet the direct and specific binding of the VxPxtargeting motif to Arf4 suggests a distinct role for this

particular Arf in the generation of RTCs. The targeting VxPx motif binds Arf4 and recruits it to the TGN, leading to assembly of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and an Arf-GAP/ effector ASAP1. The localization of the ciliary targeting complex in photoreceptors is illustrated in Figure 1. ASAP1 catalyzes phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent GTP hydrolysis on Arf4. Transgenic frogs expressing an Arf4 mutant impaired in ASAP1mediated GTP hydrolysis, display dysfunctional rhodopsin trafficking and cytoskeletal and morphological defects, resulting in retinal degeneration. FIP3, which binds Arf4, also forms a ternary complex with Rab11 and ASAP1 and stimulates Arf GAP activity of ASAP1. Emerging evidence points to the role of ASAP1 and FIP3 as a functional module that provides temporally and spatially restricted hydrolysis of GTP bound to Arf4 at the TGN. Since ASAP1 and FIP3 act as homodimers, they may oligomerize to form a protein coat that regulates ciliary targeting, a specialized form of the TGN-to-plasma membrane trafficking.

Rhodopsin provides the spatial control for the ciliary targeting module by recruiting Arf4 to the carrier budding sites at the TGN through its VxPx targeting signal. Surprisingly, the VxPx motif is not unique to rhodopsin, but is present in other membrane proteins targeted to primary cilia such as polycystins 1 and 2, and the cyclic nucleotide-gated channel CNGB1b subunit. The VxPx from polycystin-2 also binds Arf4, suggesting that the targeting complex recruited through Arf4 is a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

SNAREs and their Regulators in Rhodopsin Trafficking

In addition to GTPases and their effectors, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are major components of the intracellular machinery responsible for targeted membrane delivery. SNAREs are considered to be directly involved in membrane fusion. After the tethering step, SNAREs are activated on the opposing donor and target membranes to form a complex that bridges the two membranes and brings them into close proximity to initiate fusion. Although SNARE pairing alone is not sufficient to determine the specificity of organelle fusion, cognate SNAREs are correctly paired in biological membranes, based on proofreading and polarized distribution leading to their relative enrichment at the appropriate fusion sites. Rabs also function by concentrating and activating SNAREs, accessory proteins and lipids, at the sites of membrane fusion and are thus required for carrier docking and fusion with the target organelle.

SNARE complexes are generally composed of a fourhelical bundle bridging opposing membranes and bringing them into close proximity to initiate fusion. Fusion with the plasma membrane requires formation of a complex between syntaxins (Qa SNAREs) and VAMPs (R-SNAREs), each contributing one helix to the fourhelix SNARE bundle, and Qbc SNAREs, either neuronal SNAP-25 or non-neuronal SNAP-23, which provide two helices to the central layer of the core complex. SNAREs are targeted to appropriate membrane domains based on specific sequences. The polarized distribution of Qa SNAREs is likely to contribute additional specificity of membrane targeting by promoting fusion with only certain target membranes. Recent evidence suggests that the local lipid environment, particularly phospholipids enriched in omega-3 and omega-6 fatty acids, also contributes to regulate SNARE function.

The membrane fusion event through which RTCs deliver rhodopsin to the cilium is mediated by a SNARE complex. Syntaxin 3 and SNAP-25 are the Q-SNAREs for the fusion of incoming RTCs with the RIS plasma membrane and, therefore, regulators of ROS biogenesis in photoreceptors. The distribution of these SNAREs in photoreceptors is illustrated in Figure 2. Remarkably, omega-3 DHA enhances syntaxin 3 incorporation into SNARE complexes at RTC fusion sites and promotes ciliary membrane expansion and ROS biogenesis. Microtubules direct the restricted distribution of syntaxin 3, consistent with the membrane cytoskeleton playing an essential role in

concentrating RTC fusion regulators around the cilium. Syntaxin 3 is the major partner for SNAP-25 in photoreceptor cells; however, SNAP-25 pairing with syntaxin 1A, or 1B, may regulate a distinct trafficking pathway in the RIS. Interestingly, Syntaxin 3 is also found at the base of mouse ROS, suggesting an additional role for this SNARE in rodent rods.

ROS is a Modified Primary Cilium

Almost all cells possess primary cilia that house an array of signal transduction modules. Long underappreciated, the cilium has recently received a great deal of attention due to the ciliary involvement in a wide range of human diseases, including retinal degeneration, polycystic kidney disease (PKD), Bardet–Biedl syndrome (BBS), and neural tube defects. Many cilium disease proteins were detected in the mouse photoreceptor ciliary proteome. Ciliary involvement in a wide range of retinal diseases has come into sharp focus in the past several years, with the molecular mechanism underlying these diseases being rapidly elucidated. Several human syndromes, including Senior–Loken syndrome, Jeune syndrome, and BBS, are also characterized by both cystic kidneys and retinal degeneration, which are often found in combination with skeletal defects or other abnormalities such as obesity, polydactyly, hypogenitalism, and developmental delay that might also be caused by defects in cilia. The organization of the small GTPases,

SNAREs and their regulators involved in the ciliary targeting of rhodopsin is schematically illustrated in Figure 3.

Twelve BBS genes have been identified. A complex composed of seven BBS proteins, the BBSome, localizes to the base of the cilium and is required for ciliogenesis. BBS3, which is not a part of BBSome encodes the Arf family GTPase Arl6. Strikingly, Rabin8, the GDP/GTP exchange factor that activates Rab8, localizes to the basal body and contacts the BBSome. In cultured epithelial cells, activated Rab8 enters the primary cilium and promotes extension of the ciliary membrane. This explains the accumulation of RTCs below the cilium in photoreceptors expressing mutant Rab8. Strikingly, activated Rab8, in its GTP-bound form interacts with another centrosomal/ciliary protein CEP290/BBS14/NPHP6, which is not a part of the BBSome. Thus, BBS may be caused by defects in Rab8-mediated vesicular transport to the cilium.

An extraordinary array of retinopathy-associated ciliary proteins includes X-linked RP1, which is localized to the proximal cilium and involved in disk morphogenesis, and RP2, which was recently identified as a GAP for the

Arf family GTPase Arl3 that is involved in kidney and photoreceptor development. A major player in ciliary morphogenesis is retinitis pigmentosa GTPase regulator (RPGR), which is homologous to RCC1, the nucleotide exchange factor for the small GTPase Ran. Mutations in the retina-specific ORF15 isoform of RPGR (RPGR (ORF15)) were found in X-linked RP3, which is associated with 10–20% of RP. RPGR appears to be a part of a ciliary and basal body protein network that, when disrupted, can result in Leber congenital amaurosis, Senior–Loken syndrome, nephronophthisis, or Joubert syndrome. RPGR (ORF15) co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin. RPGRIP1, which is affected in patients with LCA, anchors RPGR to the photoreceptor connecting cilium and participates in disk morphogenesis. RPGR also interacts with calmodulin and nephrocystin-5, a ciliary IQ domain protein, which is mutated in Senior–Loken syndrome, and with the centrosomal/ciliary protein CEP290/BBS14/NPHP6, which is truncated in earlyonset retinal degeneration in the rd16 mouse. In addition,

mutations in the gene encoding the basal body protein RPGRIP1L (RPGRIP-like), a nephrocystin-4 interactor, cause Joubert syndrome.

Another ciliary and basal body protein network linked to myosin VIIa is disrupted in human USH, the most frequent cause of combined deafness–blindness. USH is genetically heterogeneous with three clinical types, USH1–3. The scaffold protein harmonin (USH1C) integrates USH1 and USH2 molecules into protein networks. The Usher protein network is organized by the scaffold proteins SANS (USH1G), which provides a linkage to the microtubule transport machinery, and whirlin (USH2D), which anchors USH2Ab and very large G-protein- coupled receptor 1b (VLGR1b). Remarkably, the USH protein network is also a part of the periciliary ridge complex (PRC), a specialized membrane domain for docking and fusion of RTCs in Xenopus photoreceptors. Finally, the Usher protein network is linked to the Crumbs polarity complex in the retina and mutations in Crumbs cause retinitis pigmentosa (RP12).

Conclusions and Summary

Numerous diseases arise from defects in proteins that participate in membrane protein trafficking, vectorial transport, and assembly of outer segment membranes. Thus, maintenance of photoreceptor cell polarity is of utmost importance for their health and survival, and ultimately for vision.

See also: Genetic Dissection of Invertebrate Phototransduction; The Photoreceptor Outer Segment as a Sensory Cilium; The Physiology of Photoreceptor Synapses and Other Ribbon Synapses; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Retinal Degeneration through the Eye of the Fly; Rod and Cone Photoreceptor Cells: Inner and Outer Segments; Rod and Cone Photoreceptor Cells: Outer Segment Membrane Renewal; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration; Xenopus laevis as a Model for Understanding Retinal Diseases.

Further Reading

Cai, H., Reinisch, K., and Ferro-Novick, S. (2007). Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Developmental Cell 12: 671–682.

Deretic, D., Schmerl, S., Hargrave, P. A., Arendt, A., and McDowell, J. H. (1998). Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA. Proceedings of the National Academy of Sciences of the United States of America 95: 10620–10625.

Deretic, D., Williams, A. H., Ransom, N., et al. (2005). Rhodopsin C-terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ARF4. Proceedings of the National Academy of Sciences of the United States of America 102: 3301–3306.

Gillingham, A. K. and Munro, S. (2007). The small G proteins of the Arf family and their regulators. Annual Review of Cell and Developmental Biology 23: 579–611.

Green, E. S., Menz, M. D., LaVail, M. M., and Flannery, J. G. (2000). Characterization of rhodopsin mis-sorting and constitutive activation in a transgenic rat model of retinitis pigmentosa. Investigative Ophthalmology and Visual Science 41: 1546–1553.

Leroux, M. R. (2007). Taking vesicular transport to the cilium. Cell 129: 1041–1043.

Malsam, J., Kreye, S., and Sollner, T. H. (2008). Membrane fusion: SNAREs and regulation. Cellular and Molecular Life Sciences 65: 2814–2832.

Mazelova, J., Astuto-Gribble, L., Inoue, H., et al. (2009). Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4.

EMBO Journal 28: 183–192.

Mazelova, J., Ransom, N., Astuto-Gribble, L., Wilson, M. C., and Deretic, D. (2009). Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid (DHA), controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments. Journal of Cell Science 122: 2003–2013.

Moritz, O. L., Tam, B. M., Hurd, L. L., et al. (2001). Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. Molecular Biology of the Cell 12: 2341–2351.

Nachury, M. V., Loktev, A. V., Zhang, Q., et al. (2007). A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129: 1201–1213.

Papermaster, D. S., Schneider, B. G., and Besharse, J. C. (1985). Vesicular transport of newly synthesized opsin from the Golgi apparatus toward the rod outer segment. Ultrastructural immunocytochemical and autoradiographic evidence in Xenopus retinas. Investigative Ophthalmology and Visual Science 26: 1386–1404.

Shi, G., Concepcion, F. A., and Chen, J. (2004). Targeting od visual pigments to rod outer segment in rhodopsin knockout mice. In: Williams, D. S. (ed.) Photoreceptor Cell Biology and Inherited Retinal Degenerations, pp. 93–109. Singapore: World Scientific Publishing.

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Wandinger-Ness, A. and Deretic, D. (2008). Rab8a. UCSD-Nature Molecule Pages. Nature Publishing Group. doi:10.1038/mp. a001997.001901.

Relevant Websites

http://www.retina-international.com – Retina International. http://www.signaling-gateway.org – The UCSD-Nature Signaling

Gateway.

http://www.sph.uth.tmc.edu/RetNet/ – Retinal Information Network. http://webvision.med.utah.edu – WEBVISION: The organization of the

retina and visual system.