Abstract
PURPOSE: Photoreceptors are highly polarized sensory neurons containing a modified cilium known as the outer segment. This cilium has a rootlet that spans the length of the metabolically active inner segment and anchors the outer segment to the remainder of the photoreceptor. The full function and reasons for such a long rootlet in photoreceptors are not well understood. To gain deeper insight, we characterized the membrane associated with the rootlet. METHODS: Proteomic analysis was performed on immunopurified wild-type (WT) and P23H rhodopsin knock-in mouse retina. Images of mouse and human retina were acquired by transmission electron microscopy and electron tomography and protein localization in mouse retina determined by immunofluorescence and immunoelectron microscopy. RESULTS: In homozygous P23H knock-in mouse retinas, misfolded rhodopsin retained in the endoplasmic reticulum (ER) prior to degradation was found to be closely associated with rootletin and mitochondrial proteins. This observation helped reveal that the ER forms extensive interactions with the rootlet, running alongside it throughout the inner segment. Furthermore, the ER branches from the rootlet to make contact with mitochondria, Golgi, and the plasma membrane. Human rod photoreceptors had similar rootlet:ER interactions within the proximal inner segment, but differed from mouse, as the rootlet within the distal inner segment mainly interacted with mitochondria. CONCLUSIONS: These findings suggest that the rootlet plays a critical role in organizing intracellular architecture by serving as a kind of "scaffold" that supports ER positioning and allowing it to branch and form membrane contact sites with other cellular membranes.