The route of the visual receptor rhodopsin along the cilium

Abstract

The photoreceptor outer segment is the most elaborate primary cilium, containing large amounts of rhodopsin (RHO) in disk membranes that grow from a connecting cilium. The movement of RHO along the connecting cilium precedes formation of the disk membranes. However, the route that RHO takes has not been clearly determined; some reports suggest that it follows an intracellular, vesicular route along the axoneme, possibly as an adaptation for the high load of delivery or the morphogenesis of the disk endomembranes. We addressed this question by studying RHO in cilia of IMCD3 cells and mouse rod photoreceptors. In IMCD3 cilia, fluorescence recovery after photobleaching (FRAP) experiments with fluorescently tagged RHO supported the idea of RHO motility in the ciliary plasma membrane and was inconsistent with the hypothesis of RHO motility within the lumen of the cilium. In rod photoreceptors, FRAP of RHO-EGFP was altered by externally applied lectin, supporting the idea of plasmalemmal RHO dynamics. Quantitative immunoelectron microscopy corroborated our live-cell conclusions, as RHO was found to be distributed along the plasma membrane of the connecting cilium, with negligible labeling within the axoneme. Taken together, the present findings demonstrate RHO trafficking entirely via the ciliary plasma membrane. This article has an associated First Person interview with the first author of the paper.

Retinal Phenotype in the rd9 Mutant Mouse, a Model of X-Linked RP

“…While the outer segment of photoreceptors is tightly packed with membranes and proteins necessary to phototransduction, the inner segment acts as biosynthetic factory to produce needed proteins and fatty acids. The continuous flow of these fundamental factors to the outer segments relies on a highly specialized connecting cilium (Besharse et al, 1977; Wolfrum and Schmitt, 2000; Burgoyne et al, 2015; Chadha et al, 2019) and it is not surprising that almost one quarter of known photoreceptor degeneration-causing genes are involved in the function of this organelle (RetNet, the Retinal Information Network) 1 . The Retinitis Pigmentosa GTPase Regulator (RPGR) is located on the X chromosome and, together with its interactome (Zhang et al, 2019), plays a critical role for connecting cilium function (Megaw et al, 2015).…”

The single pass membrane protein MRAP2 regulates energy homeostasis by promoting primary cilia localization of the G protein-coupled receptor MC4R

“…However, the necessity of an accessory protein like MRAP2 for ciliary trafficking of specific GPCRs was not considered, suggesting that these screens may have missed a number of ciliary GPCRs, including MC4R 41,42 . Whether other ciliary GPCRs use accessory proteins for ciliary trafficking is an open question, but is hinted at by studies demonstrating that proteins, such as Rhodopsin, localize robustly to cilia in their native cell types, but less well in heterologous ciliated cells 43 .…”

Compartmentalization of Photoreceptor Sensory Cilia

“…A recent study has shown that the CTS within the IC3 loop of SSTR3 provides strong binding to a recombinant BBSome core complex, but that the IC3 loop of 5-HT-6 receptor, which is similar to that of rhodopsin, provided only weak interaction (Klink et al, 2017 ). This weak interaction, as well as the presence of an FR motif in rhodopsin helix 8, which also has weak affinity to the BBSome (Klink et al, 2017 ; Yang et al, 2020 ), may support the low presence of rhodopsin in primary cilia (Trivedi and Williams, 2010 ; Trivedi et al, 2012 ; Wang et al, 2012 ; Geneva et al, 2017 ; Chadha et al, 2019 ).…”

“…Moreover, recent careful EM studies have clearly shown nascent discs to be open to the extracellular milieux (Burgoyne et al, 2015 ; Ding et al, 2015 ; Volland et al, 2015 ). Finally, immunogold labeling experiments have shown rhodopsin to be located in the ciliary membrane of mouse photoreceptors with little in the lumen of the cilium (Wolfrum and Schmitt, 2000 ; Burgoyne et al, 2015 ; Chadha et al, 2019 ).…”