Visual arrestin 1 (Arr1) is an essential protein for termination of the light response in photoreceptors. While mammalian Arr1s form dimers and tetramers at physiological concentrations in vitro, oligomerization in other vertebrates has not been studied. Here we examine self-association of Arr1 from two amphibian species, Xenopus laevis (xArr1) and Ambystoma tigrinum (salArr1). Sedimentation velocity analytical ultracentrifugation showed that xArr1 and salArr1 oligomerization is limited to dimers. The KD for dimer formation was 53 μM for xArr1 and 44 μM for salArr1, similar to the 69 μM KD for bovine Arr1 (bArr1) dimers. Mutations of orthologous amino acids important for mammalian Arr1 oligomerization had no impact on xArr1 dimerization. Crystallography showed that the fold of xArr1 closely resembles that of bArr1 and crystal structures in different space groups revealed two potential xArr1 dimer forms: a symmetric dimer with a C-domain interface (CC dimer), resembling the bArr1 solution dimer, and an asymmetric dimer with an N-domain/C-domain interface. Mutagenesis of residues predicted to interact in either of these two dimer forms yielded modest reduction in dimer affinity, suggesting that the dimer interfaces compete or are not unique. Indeed, small-angle X-ray scattering and protein painting data were consistent with a symmetric anti-parallel solution dimer (AP dimer) distinct from the assemblies observed by crystallography. Finally, a computational model evaluating xArr1 binding to compartment-specific partners and partitioning based on heterogeneity of available cytoplasmic spaces shows that Arr1 distribution in dark-adapted photoreceptors is largely explained by the excluded volume effect together with tuning by oligomerization.

The operation of photoreceptors as sensory neurons critically relies on compartmentalization of key functions into three major cellular regions. The ciliary outer segment contains the phototransduction machinery, the inner segment/cell body houses protein expression and processing, and the pre-synapse contains the machinery responsible for relaying photoreceptor electrical activity to downstream neurons. The mechanisms by which proteins that subserve these functions are compartmentalized are the subject of intense study.

A major unsolved question in vertebrate photoreceptor biology is the mechanism of rhodopsin transport to the outer segment. In rhodopsin-like class A G protein-coupled receptors, hydrophobic interactions between C -terminal alpha-helix 8 (H8), and transmembrane alpha-helix-1 (TM1) have been shown to be important for transport to the plasma membrane, however whether this interaction is important for rhodopsin transport to ciliary rod outer segments is not known. We examined the crystal structures of vertebrate rhodopsins and class A G protein-coupled receptors and found a conserved network of predicted hydrophobic interactions. In Xenopus rhodopsin (xRho), this interaction corresponds to F313, L317, and L321 in H8 and M57, V61, and L68 in TM1. To evaluate the role of H8-TM1 hydrophobic interactions in rhodopsin transport, we expressed xRho-EGFP where hydrophobic residues were mutated in Xenopus rods and evaluated the effi- ciency of outer segment enrichment. We found that substituting L317 and M57 with hydrophilic residues had the strongest impact on xRho mislocalization. Substituting hydrophilic amino acids at positions L68, F313, and L321 also had a significant impact. Replacing L317 with M resulted in significant mislocalization, indicating that the hydrophobic interaction between residues 317 and 57 is exquisitely sensitive. The corresponding experiment in bovine rhodopsin expressed in HEK293 cells had a similar effect, showing that the H8-TM1 hydrophobic network is essential for rhodopsin transport in mammalian species. Thus, for the first time, we show that a hydrophobic interaction between H8 and TM1 is critical for efficient rhodopsin transport to the vertebrate photoreceptor ciliary outer segment.

Retinal photoreceptors are neurons that convert dynamically changing patterns of light into electrical signals that are processed by retinal interneurons and ultimately transmitted to vision centers in the brain. They represent the essential first step in seeing without which the remainder of the visual system is rendered moot. To support this role, the major functions of photoreceptors are segregated into three main specialized compartments-the outer segment, the inner segment, and the pre-synaptic terminal. This compartmentalization is crucial for photoreceptor function-disruption leads to devastating blinding diseases for which therapies remain elusive. In this review, we examine the current understanding of the molecular and physical mechanisms underlying photoreceptor functional compartmentalization and highlight areas where significant knowledge gaps remain.

Functional compartmentalization of cells is a universal strategy for segregating processes that require specific components, undergo regulation by modulating concentrations of those components, or that would be detrimental to other processes. Primary cilia are hair-like organelles that project from the apical plasma membranes of epithelial cells where they serve as exclusive compartments for sensing physical and chemical signals in the environment. As such, molecules involved in signal transduction are enriched within cilia and regulating their ciliary concentrations allows adaptation to the environmental stimuli. The highly efficient organization of primary cilia has been co-opted by major sensory neurons, olfactory cells and the photoreceptor neurons that underlie vision. The mechanisms underlying compartmentalization of cilia are an area of intense current research. Recent findings have revealed similarities and differences in molecular mechanisms of ciliary protein enrichment and its regulation among primary cilia and sensory cilia. Here we discuss the physiological demands on photoreceptors that have driven their evolution into neurons that rely on a highly specialized cilium for signaling changes in light intensity. We explore what is known and what is not known about how that specialization appears to have driven unique mechanisms for photoreceptor protein and membrane compartmentalization.

In neurons, peripheral membrane proteins are enriched in subcellular compartments, where they play key roles, including transducing and transmitting information. However, little is known about the mechanisms underlying their compartmentalization. To explore the roles of hydrophobic and electrostatic interactions, we engineered probes consisting of lipidation motifs attached to fluorescent proteins by variously charged linkers and expressed them in Xenopus rod photoreceptors. Quantitative live cell imaging showed dramatic differences in distributions and dynamics of the probes, including presynapse and ciliary OS enrichment, depending on lipid moiety and protein surface charge. Opposing extant models of ciliary enrichment, most probes were weakly membrane bound and diffused through the connecting cilium without lipid binding chaperone protein interactions. A diffusion-binding-transport model showed that ciliary enrichment of a rhodopsin kinase probe occurs via recycling as it perpetually leaks out of the ciliary OS. The model accounts for weak membrane binding of peripheral membrane proteins and a leaky connecting cilium diffusion barrier.

Physical properties of primary cilia membranes in living cells were examined using two independent, high-spatiotemporal-resolution approaches: fast tracking of single quantum dot–labeled G protein–coupled receptors and a novel two-photon super-resolution fluorescence recovery after photobleaching of protein ensemble. Both approaches demonstrated the cilium membrane to be partitioned into corralled domains spanning 274 ± 20 nm, within which the receptors are transiently confined for 0.71 ± 0.09 s. The mean membrane diffusion coefficient within the corrals, Dm1 = 2.9 ± 0.41 µm2/s, showed that the ciliary membranes were among the most fluid encountered. At longer times, the apparent membrane diffusion coefficient, Dm2 = 0.23 ± 0.05 µm2/s, showed that corral boundaries impeded receptor diffusion 13-fold. Mathematical simulations predict the probability of G protein–coupled receptors crossing corral boundaries to be 1 in 472. Remarkably, latrunculin A, cytochalasin D, and jasplakinolide treatments altered the corral permeability. Ciliary membranes are thus partitioned into highly fluid membrane nanodomains that are delimited by filamentous actin.

Arrestins play important roles in GPCR signaling and desensitization. The photoreceptor specific visual arrestin (Arr1) is a soluble protein with no known post-translational modifications, that is differently distributed throughout light and dark-adapted photoreceptor. Second only to its target GPCR, rhodopsin in concentration, mechanisms that underlie the translocation of such an abundant protein are not understood. Arr1 is predicted to self-associate up to tetramers across multiple mammalian species, varying mostly in the KD of the dimer and tetramer forms. One hypothesis on the localization of Arr1 in the cell body of dark-adapted photoreceptors is that the Arr1 tetramer is partitioned there due to differences in densities of subcellular structures in the two compartments and the impact of steric volume exclusion on the accessible volumes. To investigate whether this self-associative behavior applies across a broader range of species and other model organisms, we examined Xenopus laevis Arr1 (xArr1) which bears ∼68% sequence identity to mammalian Arr1s. Homology modeling based on the bovine crystal structure suggests that it is likely to have a very similar tertiary structure. Residues of mammalian Arr1 hypothesized to be at dimer and tetramer interfaces by their ability to affect the extent of oligomerization are mostly conserved in X. laevis as well, suggesting xArr1 would likely follow the same self-association pattern. We performed analytical ultracentrifugation experiments on xArr1 as well as bovine Arr1 (bArr1) for comparison. Our data for bArr1 agrees well with KDs from published studies, and the KD dimer for xArr1 fits well into the range published for human, mouse, and bovine Arr1. Surprisingly, at concentrations up to 210 micromolar, our results show xArr1 as monomer and dimer only, with no evidence of higher order oligomers. The mechanism underlying this difference is under investigation.

Current understanding of the mechanisms underlying G-Protein Coupled Receptor (GPCR) transport within primary cilium is limited and several competing models exist, including motor-driven transport, local binding and free diffusion. Our analysis of the mean square displacement (MSD) of single GPCRs within ciliary membranes as a function of time step magnitude showed predominant mode of transport is diffusion with transient confinement to membrane sub-regions. The mechanism of this confinement is not well understood, and we hypothesize that actin filaments delimit the membrane sub-regions. We performed super-resolution high speed imaging of single GPCRs transfected into murine inner-medullary collecting duct (IMCD3) cells using quantum-dots. Latrunculin A, Cytochalaisin D, and Jasplakinolide was used to alter actin dynamics in primary cilium. The MSD(tau) analysis revealed free and fast local diffusion, followed by confinement to membrane sub-regions, slowing diffusion ∼15 fold. The average diffusion coefficient of the free local diffusion was [2.53 ± 0.42 micro m2 s−1], which is an order of magnitude higher than previous measurements of GPCR diffusion coefficient inside cilium. The average diffusion coefficient of the slow confined diffusion was [0.18 ± 0.03 mm2 s−1]. The size of the corral is calculated to be approximately 260nm. Disruption of actin by Latrunculin-A and Cytochalasin D resulted in increased diffusion coefficient and MSD. Latrunculin A treatment resulted in a 1.53±0.19 fold increase in the diffusion coefficient above vehicle control. Enhancing actin polymerization with Jasplakinolide significantly decreased the diffusion coefficient and MSD(tau). Fast imaging and the MSD analysis following altered actin dynamics demonstrates that the predominant modes of GPCR movement within the ciliary compartment is free local diffusion in highly fluid membrane, followed by transient confinement to membrane sub-regions delimited by actin. Ongoing studies aim to understand the physiological role of the ciliary membrane compartmentalization.