The P23H rhodopsin mouse model reveals a novel interaction between the endoplasmic reticulum and connecting cilium rootlet within photoreceptors

Purpose: Photoreceptors are highly polarised 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 characterised the membrane associated with the rootlet. Methods: Proteomic analysis was performed on immunopurified wild-type and P23H rhodopsin knock-in mouse retina. Images of mouse and human retina were acquired by transmission electron microscopy and electron tomography and protein localisation in mouse retina determined by immunofluorescence and immuno electron 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 organising intracellular architecture by serving as a kind of ‘scaffold’ that supports ER positioning and allowing it to branch and form membrane contact sites other cellular membranes.