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The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare −/− mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

It is important to elucidate how retinal stimulation leads to retinal protection and dysfunction. The current study aimed to identify factors that are up- and downregulated in the retinas of streptozotocin (STZ)-induced diabetic rats with acute retinal dysfunction. Retinal function was measured and changes in protein expressions were determined using electroretinograms (ERGs) and liquid chromatography/mass spectroscopy-based shotgun proteomics, respectively. The results revealed that the plasma glucose levels of STZ rats were markedly higher when compared with normal rats. Furthermore, levels of a-waves, b-waves and oscillatory potential amplitudes on ERGs in STZ rats were decreased compared with healthy animals. With use of shotgun proteomics, 391 proteins were identified in the retinas of normal rats and 541 proteins were found in the retinas of STZ rats. Of the 560 proteins identified in rat retinas, 372 (66.4%) were present in both normal and STZ rats. Of these, 19 (3.39%) were unique to normal rats and 169 (30.1%) were unique to STZ rats. Gene Ontology analysis was performed on the candidate proteins that were differentially regulated in the retinas of STZ rats and focused on those classified as 'protein binding', which serve important roles in retinal neurodegeneration. The results revealed an excessive expression of retinol-binding protein 1 (RBP1) and a negative expression of rod outer segment membrane protein 1 (Rom-1) in the retinas of STZ rats. Therefore, retinal function may be decreased with STZ-induced injury, and expressions of Rom-1 and RBP1 may be altered in the retinas of STZ rats.

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.

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations have been reported to compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkably precise arrangement of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data confirm the existence of two previously observed molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further provide evidence that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) oligomers. We suggest that together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchainforming P/rds generated abundant high-curvature OS membranes, whichwere improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions.

Vertebrate photoreceptors are among the most metabolically active cells, exhibiting a high rate of ATP consumption. This is coupled with a high anabolic demand, necessitated by the diurnal turnover of a specialized membrane-rich organelle, the outer segment, which is the primary site of phototransduction. How photoreceptors balance their catabolic and anabolic demands is poorly understood. Here, we show that rod photoreceptors in mice rely on glycolysis for their outer segment biogenesis. Genetic perturbations targeting allostery or key regulatory nodes in the glycolytic pathway impacted the size of the outer segments. Fibroblast growth factor signaling was found to regulate glycolysis, with antagonism of this pathway resulting in anabolic deficits. These data demonstrate the cell autonomous role of the glycolytic pathway in outer segment maintenance and provide evidence that aerobic glycolysis is part of a metabolic program that supports the biosynthetic needs of a normal neuronal cell type. Living cells need building materials and energy to grow and carry out their activities. Most cells in the body use sugars like glucose for these purposes. In a process known as glycolysis, cells break down glucose into molecules that are eventually converted to carbon dioxide and water to form the chemical ATP – the cellular currency for energy. Developing cells that have not yet fully specialized, and rapidly dividing cells, like cancer cells, consume large amounts of glucose via aerobic glycolysis (also known as the Warburg effect) as they require high levels of energy and building materials. As cells become more specialized and divide less often, they have a reduced need for building blocks, and adjust their consumption and breakdown of glucose accordingly. One exception is the photoreceptor cells, found in the light-sensitive part of our eyes. Although these specialized cells do not divide, they still need a lot of energy and building blocks to constantly renew their light-sensing and processing structures, and to capture and convert the information from the environment into signals. Previous research has shown that the eye also uses the Warburg effect. However, until now, it was not known whether the photoreceptors or other cells in the eye carry out this form of glycolysis. Using genetic tools, Chinchore et al. analysed how the photoreceptor cells in mice used glucose. The experiments demonstrated that the photoreceptors do indeed carry out the Warburg effect. Chinchore et al. further discovered that the Warburg effect is regulated by the same key enzymes and signalling molecules that cancer cells use. This indicates that specialized cells like photoreceptors might choose to retain certain metabolic features of their precursor cells, if they need to. These findings provide new insight into how photoreceptors use glucose. The next step will be to understand how aerobic glycolysis is regulated in healthy eyes as well as in eyes that are affected by degenerating diseases, which may ultimately lead to new ways of treating blindness.

Vision begins with conformational changes in photopigments. The associated electrical signature, called an early receptor potential (ERP), in rods is limited to contribution of a small fraction of rhodopsin embedded in plasma membrane. Optoretinography (ORG), using phase-sensitive optical coherence tomography, detects nanoscale deformations of retinal cells associated with physiological processes. In previous ORG studies, focused primarily on cones, deformation related to ERP was largely obscured by osmotic swelling and long stimuli. Here, we demonstrate a robust electromechanical signature of photoisomerization in rods. A green flash induces a sub-millisecond contraction of the outer segments by hundreds of nanometers, while a subsequent UV flash reverts the activated molecules, producing an opposite response of similar magnitude. ORG surpasses the sensitivity of electrical methods by integrating the response across all the discs in rod outer segments and it opens the door to fundamental studies of visual transduction in-vivo and to more specific clinical diagnosis. Interferometric imaging based on phase-sensitive OCT in living rat eyes demonstrates electromechanical deformation of the rod outer segment associated with reversible isomerization of rhodopsin, providing a new assay for studying visual transduction.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptorspecific Stx3 knockouts [Stx3 f/f(iCre75) and Stx3 f/f(CRX-Cre) ] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

We have used recent measurements of mammalian cone light responses and voltage-gated currents to calculate cone ATP utilization and compare it to that of rods. The largest expenditure of ATP results from ion transport, particularly from removal of Na⁺ entering outer segment light-dependent channels and inner segment hyperpolarization-activated cyclic nucleotide-gated channels, and from ATP-dependent pumping of Ca2+ entering voltage-gated channels at the synaptic terminal. Single cones expend nearly twice as much energy as single rods in darkness, largely because they make more synapses with second-order retinal cells and thus must extrude more Ca2+. In daylight, cone ATP utilization per cell remains high because cones never remain saturated and must continue to export Na⁺ and synaptic Ca2+ even in bright illumination. In mouse and human retina, rods greatly outnumber cones and consume more energy overall even in background light. In primates, however, the high density of cones in the fovea produces a pronounced peak of ATP utilization, which becomes particularly prominent in daylight and may make this part of the retina especially sensitive to changes in energy availability.

Retinal detachment (RD) is the separation of the neural layer from the retinal pigmented epithelium thereby preventing the supply of nutrients to the cells within the neural layer of the retina. In vertebrates, primary photoreceptor cells consisting of rods and cones undergo daily renewal of their outer segment through the addition of disc-like structures and shedding of these discs at their distal end. When the retina detaches, the outer segment of these cells begins to degenerate and, if surgical procedures for reattachment are not done promptly, the cells can die and lead to blindness. The precise effect of RD on the renewal process is not well understood. Additionally, a time frame within which reattachment of the retina can restore proper photoreceptor cell function is not known. Focusing on rod cells, we propose a mathematical model to clarify the influence of retinal detachment on the renewal process. Our model simulation and analysis suggest that RD stops or significantly reduces the formation of new discs and that an alternative removal mechanism is needed to explain the observed degeneration during RD. Sensitivity analysis of our model parameters points to the disc removal rate as the key regulator of the critical time within which retinal reattachment can restore proper photoreceptor cell function.