An Arrestin' Discovery♦ : Arrestin-Rhodopsin Binding Stoichiometry in Isolated Rod Outer Segment Membranes Depends on the Percentage of Activated Receptors
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PROVIDER: S-EPMC3045027 | biostudies-literature | 2011 Mar
REPOSITORIES: biostudies-literature
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Project description:In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestin-Rho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.
| S-EPMC3044992 | 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 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:The light-sensing rod photoreceptor cell exhibits several adaptations in response to the lighting environment. While adaptations to short-term changes in lighting conditions have been examined in depth, adaptations to long-term changes in lighting conditions are less understood. Atomic force microscopy was used to characterize the structure of rod outer segment disc membranes, the site of photon absorption by the pigment rhodopsin, to better understand how photoreceptor cells respond to long-term lighting changes. Structural properties of the disc membrane changed in response to housing mice in constant dark or light conditions and these adaptive changes required output from the phototransduction cascade initiated by rhodopsin. Among these were changes in the packing density of rhodopsin in the membrane, which was independent of rhodopsin synthesis and specifically affected scotopic visual function as assessed by electroretinography. Studies here support the concept of photostasis, which maintains optimal photoreceptor cell function with implications in retinal degenerations.
| S-EPMC5581674 | biostudies-literature
Project description:Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H)-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. Rho(P23H)-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H)-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H)-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H), suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.
| S-EPMC3261860 | biostudies-literature
Project description:Rod photoreceptor cells initiate scotopic vision when the light receptor rhodopsin absorbs a photon of light to initiate phototransduction. These photoreceptor cells are exquisitely sensitive and have adaptive mechanisms in place to maintain optimal function and to overcome dysfunctional states. One adaptation rod photoreceptor cells exhibit is in the packing properties of rhodopsin within the membrane. The mechanism underlying these adaptations is unclear. Mouse models of congenital stationary night blindness with different molecular causes were investigated to determine which signals are important for adaptations in rod photoreceptor cells. Night blindness in these mice is caused by dysfunction in either rod photoreceptor cell signaling or bipolar cell signaling. Changes in the packing of rhodopsin within photoreceptor cell membranes were examined by atomic force microscopy. Mice expressing constitutively active rhodopsin did not exhibit any adaptations, even under constant dark conditions. Mice with disrupted bipolar cell signaling exhibited adaptations, however, they were distinct from those in mice with disrupted phototransduction. These differential adaptations demonstrate that although multiple molecular defects can lead to a similar primary defect causing disease (i.e., night blindness), they can cause different secondary effects (i.e., adaptations). The lighting environment or signaling defects present from birth and during early rearing can condition mice and affect the adaptations occurring in more mature animals. A comparison of effects in wild-type mice, mice with defective phototransduction, and mice with defective bipolar cell signaling, indicated that bipolar cell signaling plays a role in this conditioning but is not required for adaptations in more mature animals.
| S-EPMC7387220 | biostudies-literature
Project description:Rhodopsin is the light receptor in rod photoreceptor cells that initiates scotopic vision. Studies on the light receptor span well over a century, yet questions about the organization of rhodopsin within the photoreceptor cell membrane still persist and a consensus view on the topic is still elusive. Rhodopsin has been intensely studied for quite some time, and there is a wealth of information to draw from to formulate an organizational picture of the receptor in native membranes. Early experimental evidence in apparent support for a monomeric arrangement of rhodopsin in rod photoreceptor cell membranes is contrasted and reconciled with more recent visual evidence in support of a supramolecular organization of rhodopsin. What is known so far about the determinants of forming a supramolecular structure and possible functional roles for such an organization are also discussed. Many details are still missing on the structural and functional properties of the supramolecular organization of rhodopsin in rod photoreceptor cell membranes. The emerging picture presented here can serve as a springboard towards a more in-depth understanding of the topic.
| S-EPMC8364927 | 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 phototransduction cascade in vertebrate rod visual cells is initiated by the photoactivation of rhodopsin, which enables the activation of the visual G protein transducin. It is terminated by the phosphorylation of rhodopsin, followed by the binding of arrestin. Here we measured the solution X-ray scattering of nanodiscs containing rhodopsin in the presence of rod arrestin to directly observe the formation of the rhodopsin/arrestin complex. Although arrestin self-associates to form a tetramer at physiological concentrations, it was found that arrestin binds to phosphorylated and photoactivated rhodopsin at 1:1 stoichiometry. In contrast, no complex formation was observed for unphosphorylated rhodopsin upon photoactivation, even at physiological arrestin concentrations, suggesting that the constitutive activity of rod arrestin is sufficiently low. UV-visible spectroscopy demonstrated that the rate of the formation of the rhodopsin/arrestin complex well correlates with the concentration of arrestin monomer rather than the tetramer. These findings indicate that arrestin monomer, whose concentration is almost constant due to the equilibrium with the tetramer, binds to phosphorylated rhodopsin. The arrestin tetramer would act as a reservoir of monomer to compensate for the large changes in arrestin concentration in rod cells caused by intense light or adaptation.
| S-EPMC10003454 | biostudies-literature