Compartmentalization of Photoreceptor Sensory Cilia

Abstract

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.

Keywords: outer segment; peripheral membrane protein; photoreceptors; primary cilia; rhodopsin; soluble protein; trafficking; transport.

Figure 1

Evolution of ciliary photoreceptors. (A) Schematic of an ascidian tadpole cerebral ganglion photoreceptor. Reproduced from Eakin and Kuda (1971). (B) Transmission electron micrographs of cerebral ganglion photoreceptors of Amaroucium constellatum larva. In the upper panel the section is perpendicular to the membrane lamellae. The lower panel is an image from a section parallel to the lamellar membranes. Reproduced from Barnes (1971). (C) Transmission electron micrograph of rod photoreceptors from mouse retina showing the CC, apical membrane region of the IS and nascent and enclosed discs of the OS. Note dense packing of nascent discs (arrowheads) and close juxtaposition of neighboring rods. Scale bar 1 μm. Reproduced from Ding et al. (2015). (D) Schematic of an amphibian rod. OS, outer segment; IS, inner segment; S, synaptic spherule; Ax, axon; N, nucleus; M, myoid; E, ellipsoid; CP, calycal process; ND, nascent disc; CC, connecting cilium; DM, mature disc membrane; PM, plasma membrane. Cyan: apical membrane. NDs are open to the extracellular milieu and contiguous with the CC and PM. DMs are enclosed within and separate from the OS PM and each other. Modified from Maza et al. (2019).

Figure 2

Turnover of photoreceptor OSs. Stack of discs shed from a frog (Rana pipiens) rod OS, phagocytosed by a retinal pigment epithelial cell. ROS, rod OS; P, phagosome. Scale bar, 1 μm. Reproduced from Hollyfield et al. (1977).

Figure 3

Photoreceptors periciliary membrane systems. (A) Scanning electron micrographs of apical surfaces of frog (Rana pipiens) rods whose OSs where broken off, exposing the ISs. Note remnants of the CC (CC) recessed in pits at the periphery of the apical membranes (arrows). Membrane infoldings radiating from the connecting cilia are periciliary ridge complexes (PRC). Inset (2C) is a transmission electron micrograph form a section cut perpendicular to the CC. Scale bars, 2A, 2 μm; 2B, 0.5 μm; 2C, 0.5 μm. Reproduced from Peters et al. (1983). (B) Transmission electron micrograph through of a mouse rod where the plane of section bisected the CC (CC) and the basal body (BB). A deep ciliary pocket/periciliary membrane extends along one side of the CC (CC) and the nascent disc membranes (arrowhead). The opposite side of the CC abuts the neighboring photoreceptor. IS, IS; Mt, mitochondrion. Scale bare, 1 μm. Reproduced from Ding et al. (2015).

Figure 4

Structure of the photoreceptor CC. (A) Surface representations of cryo-EM tomographs subject to 9-fold sub tomograph averaging. Left, section through the ciliary axis, color bar codes distance from center. Middle, entire map. Right upper, distal cross section of the transition zone. Right lower, proximal transition zone cross section shows Y-link-like structures (cyan) projecting between the axoneme (magenta) and the ciliary membrane (yellow). (B) Diagram showing the resolution of concentric layers of the CC determined by STORM microscopy, overlaid on cryo-EM tomographic map of ciliary cross section. (C) STORM localization maps a CC co-labeled with IFT88 and BBS5. Left panel shows tubulin in bright field to delineate the CC length. Expanded images show close juxtaposition of IFT88 (cyan) and BBS5 (magenta). Right two panels show individual channels. Yellow arrows are suggested to show co-localization. Modified from Robichaux et al. (2019).

Figure 5

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.

Figure 6

The opsin Velcro model of rhodopsin enrichment in disc membranes. (A) Series of transmission EM images showing how nascent disc morphogenesis is thought to proceed. Note the close juxtaposition (~2–3 nm) of extracellular membrane leaflets between the previous and new lamellae as the membrane of the new lamella elongates. Reproduced from Steinberg et al. (1980). (B) 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.

Figure 7

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, PrBPδ 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 Maza et al. (2019).

Figure 8

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 hydrated radius (r_h) 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. (A,B) modified from Najafi et al. (2012).