The phototransduction machinery in the rod outer segment has a strong efficacy gradient.
Ontology highlight
ABSTRACT:
SUBMITTER: Mazzolini M
PROVIDER: S-EPMC4443333 | biostudies-literature | 2015 May
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The small GTPase, ADP-ribosylation factor-like 3 (ARL3), has been proposed to participate in the transport of proteins in photoreceptor cells. Moreover, it has been implicated in the pathogenesis associated with X-linked retinitis pigmentosa (XLRP) resulting from mutations in the ARL3 GTPase activating protein, retinitis pigmentosa 2 (RP2). To determine the importance of ARL3 in rod photoreceptor cells, we generated transgenic mice expressing a dominant active form of ARL3 (ARL3-Q71L) under a rod-specific promoter. ARL3-Q71L animals exhibited extensive rod cell death after post-natal day 30 (PN30) and degeneration was complete by PN70. Prior to the onset of cell death, rod photoresponse was significantly reduced along with a robust decrease in rod phosphodiesterase 6 (PDE6) and G-protein receptor kinase-1 (GRK1) levels. Furthermore, assembled phosphodiesterase-6 (PDE6) subunits, rod transducin and G-protein receptor kinase-1 (GRK1) accumulated on large punctate structures within the inner segment in ARL3-Q71L retina. Defective trafficking of prenylated proteins is likely due to sequestration of prenyl binding protein ? (PrBP?) by ARL3-Q71L as we demonstrate a specific interaction between these proteins in the retina. Unexpectedly, our studies also revealed a novel role for ARL3 in the migration of photoreceptor nuclei. In conclusion, this study identifies ARL3 as a key player in prenylated protein trafficking in rod photoreceptor cells and establishes the potential role for ARL3 dysregulation in the pathogenesis of RP2-related forms of XLRP.
| S-EPMC5062590 | biostudies-literature
Project description:Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation.
| S-EPMC4917604 | biostudies-literature
Project description:Rod outer segment (OS) morphogenesis involves assembly of flattened discs circumscribed by a hairpin-like rim, however, the role of the rim and rim proteins such as retinal degeneration slow (RDS) and its homologue rod OS membrane protein-1 (ROM-1) in this process remains unclear. Here we show that without RDS, no disc/OS formation occurs, while without rhodopsin, small OS structures form containing aligned nascent discs. In the absence of both rhodopsin and RDS, RDS-associated degeneration is slowed, and ROM-1 is stabilized and trafficked to the OS. These animals (rho-/-/rds-/-) exhibit OSs slightly better than those lacking only RDS, but still without signs of disc formation. These results clearly demonstrate that OS morphogenesis is initiated by RDS-mediated rim formation, a process ROM-1 cannot recapitulate, with subsequent disc growth mediated by rhodopsin. The critical role of RDS in this process helps explain why photoreceptors are so sensitive to varied RDS levels, and why mutations in RDS cause debilitating retinal disease.
| S-EPMC4045911 | biostudies-literature
Project description:The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.
| S-EPMC4672767 | biostudies-literature
Project description:BackgroundThe retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly.Methodology/principal findingsThe structure and function of rods from hemizygous rhodopsin knockout (R+/-) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/- rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics.Conclusions/significanceRhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/- rods to light.
| S-EPMC3360601 | biostudies-literature
Project description: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.
| S-EPMC8758146 | biostudies-literature
Project description:Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.
| S-EPMC4583372 | biostudies-literature
Project description:The vertebrate rod photoreceptor undergoes daily growth and shedding to renew the rod outer segment (ROS), a modified cilium that contains the phototransduction machinery. It has been demonstrated that ROS shedding is regulated by the light-dark cycle; however, we do not yet have a satisfactory understanding of the molecular mechanisms that underlie this regulation. Given that phototransduction relies on the hydrolysis of cGMP via phosphodiesterase 6 (PDE6), we examined ROS growth and shedding in zebrafish treated with cGMP-specific PDE inhibitors.We used transgenic zebrafish that express an inducible, transmembrane-bound mCherry protein, which forms a stripe in the ROS following a heat shock pulse and serves as a marker of ROS renewal. Zebrafish were reared in constant darkness or treated with PDE inhibitors following heat shock. Measurements of growth and shedding were analyzed in confocal z-stacks collected from treated retinas.As in dark-reared zebrafish, shedding was reduced in larvae and adults treated with the PDE5/6 inhibitors sildenafil and vardenafil but not with the PDE5 inhibitor tadalafil. In addition, vardenafil noticeably affected rod inner segment morphology. The inhibitory effect of sildenafil on shedding was reversible with drug removal. Finally, cones were more sensitive than rods to the toxic effects of sildenafil and vardenafil.We show that pharmacologic inhibition of PDE6 mimics the inhibition of shedding by prolonged constant darkness. The data show that the influence of the light-dark cycle on ROS renewal is regulated, in part, by initiating the shedding process through activation of the phototransduction machinery.
| S-EPMC5667398 | biostudies-literature
Project description:Vectors based on the adeno-associated virus (AAV) are currently the preferred tools for delivering genes to photoreceptors (PR) in small and large animals. AAVs have been applied successfully in various models of PR dystrophies. However, unknown barriers still limit AAV's efficient application in several forms of severe PR degenerations due to insufficient transgene expression and/or treated cells at the time of injection. Optimizations of PR gene therapy strategies will likely benefit from the identification of the cellular factors that influence PR transduction. Interestingly, recent studies have shown that the AAV transduction profile of PRs differs significantly between neonatal and adult mouse retinas after subretinal injection. This phenomenon may provide clues to identify host factors that influence the efficiency of AAV-mediated PR transduction. This study demonstrates that rod outer segments are critical modulators of efficient AAV-mediated rod transduction. During retinal development, rod transduction correlated temporally and spatially with the differentiation order of PRs when vectors were introduced subretinally but not when introduced intravitreally. All subretinally injected vectors had an initial preference to transduce cones in the absence of formed rod outer segments and then displayed a preference for rods as the cells matured, independently of the expression cassette or AAV serotype. Consistent with this observation, altered development of rod outer segments was associated with a strong reduction of rod transduction and an increase in the percentage of transduced cones by 2- to 2.8-fold. A similar increase of cone transduction was observed in the adult retinal degeneration 1 (rd1) retina compared to wild-type mice. These results suggest that the loss of rod outer segments in diseased retinas could markedly affect gene transfer efficiency of AAV vectors by limiting the ability of AAVs to infect dying rods efficiently. This information could be exploited for the development of more efficient AAV-based PR gene delivery procedures.
| S-EPMC5488363 | biostudies-literature
Project description:The transduction of light by retinal rods and cones is effected by homologous G-protein cascades whose rates of activation and deactivation determine the sensitivity and temporal resolution of photoreceptor signaling. In mouse rods, the rate-limiting step of deactivation is hydrolysis of GTP by the G-protein-effector complex, catalyzed by the RGS9 complex. Here, we incorporate a "Michaelis module" describing the RGS9 reaction into the conventional scheme for phototransduction and show that this augmented scheme can account precisely for the dominant recovery rate of intact rods in which RGS9 expression varies over a 20-fold range. Furthermore, by screening the parameter space of the scheme with maximum-likelihood methodology, we tested specific hypotheses about the rate constant for rhodopsin deactivation, and about the forward, reverse, and catalytic constants for RGS9-mediated G-protein deactivation. These tests reliably exclude lifetimes > approximately 50 ms for rhodopsin, and reveal that the dominant time constant of rod photoresponse recovery is 1/(V(max)/K(m)) for the RGS9 reaction, with k(cat)/K(m) approximately = 0.04 microm(2) s(-1) and k(cat) > 35 s(-1) (or K(m) > 840 microm(-2)). All together, the new kinetic scheme and analysis explain how and why RGS9 concentration matters in rod phototransduction, and they provide a framework for understanding the molecular mechanisms that rate-limit deactivation in other G-protein systems.
| S-EPMC2749774 | biostudies-literature