Functional compartmentalization of photoreceptor neurons

Abstract

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.

Keywords: Arrestin; Cilia; Membrane proteins; Photoreceptor; Rhodopsin; Trafficking.

Fig. 1. Schematic of rod and cone photoreceptors

Simplified schematics of a rod photoreceptor (left) and a cone photoreceptor (right), indicating the functional compartments. Plus and minus indicate polarity of the microtubules. Axoneme microtubules are solid blue lines, cellular microtubules are grey broken lines. Orange circles represent the adherens junctions/outer limiting membrane. Red circles indicate subapical region (SAR). CP=Calyceal process, OS = Outer segment, IS = inner segment, CC = connecting cilium, Syn = synapse. H = horizontal cell process, B = ON bipolar cell process, OFF BC = OFF bipolar cell process.

Fig. 2. Intrinsic membrane protein compartmentalization within the OS.

Schematic of a mammalian rod in the region of the CC. Intrinsic membrane proteins are thought to be delivered to the apical/periciliary membrane on rhodopsin transport carrier (RTC) vesicles, where they fuse (see text for details). Membrane proteins then enter the CC/transition zone, possibly mediated by the BBSome. Proteins destined for disc and plasma membrane transport to the lamellar membranes most likely by diffusion. Eventually the nascent disc membranes are enclosed by the plasma membrane through a mechanism that separates plasma membrane proteins, the CNG gated channel-Na⁺/Ca²⁺,K⁺ exchanger complex, from the disc proteins, rhodopsin and peripherin 2, which likely occurs during peripherin 2-Rom1-mediated disc rim expansion (see text). Note that this schematic is not meant to be an exhaustive representation of all OS membrane protein transport, or of disc morphogenesis.

Fig. 3. The Stochastic Velcro model of rhodopsin enrichment in disc membranes.

Rhodopsin density in the disc membranes is twice that in the plasma membrane, indicating that it is not efficiently separated into disc membranes. One possible explanation for this asymmetry is that rhodopsin self-associates at is extracellular N-termini. This may result in a Velcro-like coupling of the nascent disc membranes and producing a self-binding sink driving disc enrichment. Affinity would not be expected to be high for this interaction since it only produces a twofold difference in disc vs plasma membrane density. This interaction may also help drive disc morphogenesis. For simplicity, rhodopsin cis dimers (in the same membrane) are not depicted in this schematic.

Fig. 4. Role of electrostatic interactions in the compartmentalization of peripheral membrane proteins.

A. Montage of confocal images of living Xenopus rods expressing EGFP probes with indicated surface charge and lipidation motif. Note that none of the probes possess binding motifs for the lipid binding chaperone proteins, PrBPδ and Unc119. Significant OS localization of most probes shows that lipid binding chaperone proteins are not required for OS access and enrichment of peripheral membrane proteins. B. Box-whisker plots of average fluorescence in the pre-synapse divided by average fluorescence in the OS shows that positively charged probes with prenyl lipids are depleted from the OS and enriched in the pre-synapse, while probes containing myristoylation and neutral or negative charge equally distributed between compartments. A probe consisting of EGFP fused to the myristoylation motif containing N-terminal 16 amino acids of Tα, which binds to Unc119, was not significantly more OS enriched than the Myr0 probe, which does not bind Unc119, suggesting that Unc119 association alone is not sufficient for OS enrichment. The probe containing the farnesylated C-terminus of GRK1, which does bind to PrBPδ, is more strongly OS localized, thus, PrBP delta tilted the equilibrium toward OS enrichment. However, presence of the Far0 probe in the OS shows that PrBPδ is not required for OS entry. Modified from [130].

Fig. 5. Steric volume exclusion and the compartmentalization of soluble proteins in photoreceptors.

A. The distribution of soluble molecules in Xenopus rods depends on the size of the molecule. Note that the conformation of the EGFP dimers and tetramer shown are only one of many possible. B. The relationship of the ratio of the OS fluorescence to the maximum IS fluorescence scaled inversely and linearly with the estimated average hydrated radius of the molecules. This phenomenon can be explained by the asymmetrical reduction in the available aqueous volume of the differently shaped compartments caused by steric volume exclusion (i.e. loss of volume available to the center of mass of the molecule). C. For example, two interconnected boxes have the same geometric volume (V_g), but vastly different shapes. Introducing a spherical molecule reduces the geometry of both compartments, and thus the volumes accessible (V_ac) to their centers of mass of the molecule. This reduction is larger for the rectangular compartment. D. As the size of the molecules increase, the reduction in V_ac falls more steeply for the rectangular compartment. E. Since soluble molecules will equilibrate to equalize their concentrations (c) everywhere, the shape asymmetry will cause partitioning of the soluble molecules into the cubical compartment, where the total mass (m) will be higher. Panels A and B modified from [147].

Figure 6.. DBT model predicts the distribution of Arr1 in rods possessing bleached-unphosphorylated and bleached-phosphorylated rhodopsin to be nearly indistinguishable.

We calculated the distribution of Arr1 concentration in rod photoreceptors using our diffusion, binding active transport (DBT) model [130]. We used our published values for compartment specific soluble protein diffusion coefficients [28] and the dissociation constants for Arr1 binding with bleached-unphosphorylated rhodopsin or bleached-phosphorylated rhodopsin [249]. The concentration of rhodopsin in the outer segment was assumed to be 6 mM [171], and that of Arr1 to be 4.7 mM [202], relative to the disc excluded outer segment volume. Despite three orders of magnitude differences in K_d, the distribution of Arr1 was strongly outer segment biased in both bleached rods. This can be explained by the high concentration of Arr1 and rhodopsin relative to the K_ds for respective bleached rhodopsins and the lack of binding sites in the inner segment near the concentration of Arr1. Despite the strong outer segment enrichment of Arr1 in the case of bleached-unphosphorylated rhodopsin, the outer segment mobility remained reasonably high due to higher off rate. The distribution of Arr1 in the dark-adapted rod assumed inner segment partitioning via the steric volume exclusion mechanism [147].

Fig. 7. Interactions of photoreceptor ribbon synapse proteins.

Schematic of the ribbon synapse protein interactions. Schematic based on [62, 135, 237], among others.