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The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or “discs”, located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called “incisures”. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened “disc” membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of twenty key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio amongst all twenty proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wildtype and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

Mutations in thePRPH2gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened “disc” membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous “whorls” instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENTThe light-sensitive photoreceptor outer segment contains a stack of flattened “disc” membranes that are surrounded, or “enclosed,” by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

S100A4, a member of the S100 family of multifunctional calcium-binding proteins, participates in several physiological and pathological processes. In this study, we demonstrate that S100A4 expression is robustly induced in differentiating fiber cells of the ocular lens and that S100A4(−/−) knockout mice develop late-onset cortical cataracts. Transcriptome profiling of lenses from S100A4(−/−) mice revealed a robust increase in the expression of multiple photoreceptor- and Müller glia-specific genes, as well as the olfactory sensory neuron-specific gene, S100A5. This aberrant transcriptional profile is characterized by corresponding increases in the levels of proteins encoded by the aberrantly upregulated genes. Ingenuity pathway network and curated pathway analyses of differentially expressed genes in S100A4(−/−) lenses identified Crx and Nrl transcription factors as the most significant upstream regulators, and revealed that many of the upregulated genes possess promoters containing a high-density of CpG islands bearing trimethylation marks at histone H3K27 and/or H3K4, respectively. In support of this finding, we further documented that S100A4(−/−) knockout lenses have altered levels of trimethylated H3K27 and H3K4. Taken together, our findings suggest that S100A4 suppresses the expression of retinal genes during lens differentiation plausibly via a mechanism involving changes in histone methylation.

The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment., Graphical Abstract, Highlights • The unique proteome of the photoreceptor outer segment plasma membrane is identified. • TMEM67, TMEM237, and embigin are novel unique components of this membrane. • Embigin in this membrane is associated with the monocarboxylate transporter MCT1. • The photoreceptor outer segment likely facilitates lactate transport., In Brief The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate photoreceptor cells plays diverse roles in supporting photoreceptor function and health. Protein correlation profiling of this membrane revealed a surprisingly small number of unique protein components. Among them are TMEM67 and TMEM237, whose mutations are associated with various syndromic ciliopathies, and embigin found to be associated with the monocarboxylate transporter MCT1. The MCT1–embigin complex likely facilitates lactate transport through this cellular compartment.

The light-sensitive outer segment organelle of the vertebrate photoreceptor cell is a modified cilium filled with hundreds of flattened 'disc' membranes that provide vast light-absorbing surfaces. The outer segment is constantly renewed with new discs added at its base every day. This continuous process is essential for photoreceptor viability. In this review, we describe recent breakthroughs in the understanding of disc morphogenesis, with a focus on the molecular mechanisms responsible for initiating disc formation from the ciliary membrane. We highlight the discoveries that this mechanism evolved from an innate ciliary process of releasing small extracellular vesicles, or ectosomes, and that both disc formation and ectosome release rely on the actin cytoskeleton.

Using structured illumination microscopy and photoconvertible tubulin in zebrafish photoreceptors, it is shown that microtubules move together during myoid elongation, a dark adaptive process in cone photoreceptors. Additionally, cytoplasmic dynein-1, localized at the base of the elongating myoid, mediates this unidirectional movement of microtubules., Teleosts and amphibians exhibit retinomotor movements, morphological changes in photoreceptors regulated by light and circadian rhythms. Cone myoid elongation occurs during dark adaptation, leading to the positioning of the cone outer segment closer to the retinal pigment epithelium. Although it has been shown that microtubules are essential for cone myoid elongation, the underlying mechanism has not been established. In this work, we generated a transgenic line of zebrafish expressing a photoconvertible form of α-tubulin (tdEOS-tubulin) specifically in cone photoreceptors. Using superresolution structured illumination microscopy in conjunction with both pharmacological and genetic manipulation, we show that cytoplasmic dynein-1, which localizes to the junction between the ellipsoid and myoid, functions to shuttle microtubules from the ellipsoid into the myoid during the course of myoid elongation. We propose a novel model by which stationary complexes of cytoplasmic dynein-1 are responsible for the shuttling of microtubules between the ellipsoid and myoid is the underlying force for the morphological change of myoid elongation.

Retinitis pigmentosa is a retinal degenerative disease that leads to blindness through photoreceptor loss. Rhodopsin is the most frequently mutated protein in this disease. While many rhodopsin mutations have well-understood consequences that lead to cell death, the disease association of several rhodopsin mutations identified in retinitis pigmentosa patients, including F220C and F45L, has been disputed. In this study, we generated two knockin mouse lines bearing each of these mutations. We did not observe any photoreceptor degeneration in either heterozygous or homozygous animals of either line. F220C mice exhibited minor disruptions of photoreceptor outer segment dimensions without any mislocalization of outer segment proteins, whereas photoreceptors of F45L mice were normal. Suction electrode recordings from individual photoreceptors of both mutant lines showed normal flash sensitivity and photoresponse kinetics. Taken together, these data suggest that neither the F220C nor F45L mutation has pathological consequences in mice and, therefore, may not be causative of retinitis pigmentosa in humans.

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a “lamellipodium-like” mechanism.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood–retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using theMfsd2aknock-out mouse as animal model. Our lipidome analyses ofMfsd2a−/−retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated inMfsd2a−/−retinas.Mfsd2a−/−retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructureMfsd2a−/−photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENTPhospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood–retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

Fluorescent proteins are commonly used to label target proteins in live cells. However, the conventional approach based on covalent fusion of targeted proteins with fluorescent protein probes is limited by the slow rate of fluorophore maturation and irretrievable loss of fluorescence due to photobleaching. Here, we report a genetically encoded protein labeling system utilizing transient interactions of small, 21–28 residues-long helical protein tags (K/E coils, KEC). In this system, a protein of interest, covalently tagged with a single coil, is visualized through binding to a cytoplasmic fluorescent protein carrying a complementary coil. The reversible heterodimerization of KECs, whose affinity can be tuned in a broad concentration range from nanomolar to micromolar, allows continuous exchange and replenishment of the tag bound to a targeted protein with the entire cytosolic pool of soluble fluorescent coils. We found that, under conditions of partial illumination of living cells, the photostability of labeling with KECs exceeds that of covalently fused fluorescent probes by approximately one order of magnitude. Similarly, single-molecule localization microscopy with KECs provided higher labeling density and allowed a much longer duration of imaging than with conventional fusion to fluorescent proteins. We also demonstrated that this method is well suited for imaging newly synthesized proteins, because the labeling efficiency by KECs is not dependent on the rate of fluorescent protein maturation. In conclusion, KECs can be used to visualize various target proteins which are directly exposed to the cytosol, thereby enabling their advanced characterization in time and space.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

BackgroundKIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown.ResultsUsing transgenic expression in both mammalian cells and zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization to cilia. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding.ConclusionsTaken together, our data support a model in which phosphorylation of KIF17 promotes its ciliary localization. In cone photoreceptor outer segments, this promotes disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.

Zebrafish morphants of osm-3/kif17, a kinesin-2 family member and intraflagellar transport motor, have photoreceptor outer segments that are dramatically reduced in number and size. However, two genetic mutant lines, osm-3/kif17sa0119 and osm-3/kif17sa18340, reportedly lack any observable morphological outer segment defects. In this work, we use TALENs to generate an independent allele, osm-3/kif17mw405, and show that both osm-3/kif17sa0119 and osm-3/kif17mw405 have an outer segment developmental delay in both size and density that is fully recovered by 6 days post-fertilization. Additionally, we use CRISPRs to generate cos2/kif7mw406, a mutation in the kinesin-4 family member cos2/kif7 that has been implicated in controlling ciliary architecture and Hedgehog signaling to test whether it may be functioning redundantly with osm-3/kif17. We show that cos2/kif7mw406 has an outer segment developmental delay similar to the osm-3/kif17 mutants. Using a three-dimensional mathematical model of outer segments, we show that while cos2/kif7mw406 and osm-3/kif17mw405 outer segments are smaller throughout the first 6 days of development, the volumetric rates of outer segment morphogenesis are not different among wild-type, cos2/kif7mw406, and osm-3/kif17mw405 after 60hpf. Instead, our model suggests that cos2/kif7mw406 and osm-3/kif17mw405 impact outer segment morphogenesis through upstream events that that are different for each motor. In the case of cos2/kif7mw406 mutants, we show that early defects in Hedgehog signaling lead to a general, non-photoreceptor-specific delay of retinal neurogenesis, which in turn causes the secondary phenotype of delayed outer segment morphogenesis. In contrast, the osm-3/kif17mw405 outer segment morphogenesis delays are linked specifically to initial disc morphogenesis of photoreceptors rather than an upstream event. Further, we show that osm-3/kif17 mutant mice also exhibit a similarly delayed outer segment development, suggesting a role for osm-3/kif17 in early outer segment development that is conserved across species. In conclusion, we show that both osm-3/kif17 and cos2/kif7 have comparable outer segment developmental delays, although through independent mechanisms.

Author(s): Sajdak, Benjamin S; Bell, Brent A; Lewis, Tylor R; Luna, Gabriel; Cornwell, Grayson S; Fisher, Steven K; Merriman, Dana K; Carroll, Joseph | Abstract: Purpose:We examined outer retinal remodeling of the euthermic and torpid cone-dominant 13-lined ground squirrel (13-LGS) retina using optical coherence tomography (OCT) imaging and histology. Methods:Retinas and corneas of living 13-LGSs were imaged during euthermic and torpid physiological states using OCT. Retinal layer thickness was measured at the visual streak from registered and averaged vertical B-scans. Following OCT, some retinas were collected immediately for postmortem histologic comparison using light microscopy, immunofluorescence, or transmission electron microscopy. Results:Compared to OCT images from euthermic retinae, OCT images of torpid retinae revealed significantly thicker inner and outer nuclear layers, as well as increases in the distances between outer retinal reflectivity bands 1 and 2, and bands 3 and 4. A significant decrease in the distance between bands 2 and 3 also was seen, alongside significant thinning of the choriocapillaris and choroid. OCT image quality was reduced in torpid eyes, partly due to significant thickening of the corneal stroma during this state. Conclusions:The torpid retina of the hibernating 13-LGS undergoes structural changes that can be detected by OCT imaging. Comparisons between in vivo OCT and ex vivo histomorphometry may offer insight to the origin of hyperreflective OCT bands within the outer retina of the cone-dominant 13-LGS.