Summary Defects in primary cilia lead to devastating disease due to their roles in sensation and developmental signaling, but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain three-dimensional maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wildtype and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.
In order to identify candidate cation channels important for retinal physiology, 28 TRP channel genes were surveyed for expression in the mouse retina. Transcripts for all TRP channels were detected by RT-PCR and sequencing. Northern blotting revealed that mRNAs for 12 TRP channel genes are enriched in the retina. The strongest signals were observed for TRPC1, TRPC3, TRPM1, TRPM3, and TRPML1, and clear signals were obtained for TRPC4, TRPM7, TRPP2, TRPV2, and TRPV4. In situ hybridization and immunofluorescence revealed widespread expression throughout multiple retinal layers for TRPC1, TRPC3, TRPC4, TRPML1, PKD1, and TRPP2. Striking localization of enhanced mRNA expression was observed for TRPC1 in the photoreceptor inner segment layer, for TRPM1 in the inner nuclear layer (INL), for TRPM3 in the INL, and for TRPML1 in the outer plexiform and nuclear layers. Strong immunofluorescence signal in cone outer segments was observed for TRPM7 and TRPP2. TRPC5 immunostaining was largely confined to INL cells immediately adjacent to the inner plexiform layer. TRPV2 antibodies stained photoreceptor axons in the outer plexiform layer. Expression of TRPM1 splice variants was strong in the ciliary body, whereas TRPM3 was strongly expressed in the retinal pigmented epithelium.
The rod cell has an extraordinarily specialized structure that allows it to carry out its unique function of detecting individual photons of light. Both the structural features of the rod and the metabolic processes required for highly amplified light detection seem to have rendered the rod especially sensitive to structural and metabolic defects, so that a large number of gene defects are primarily associated with rod cell death and give rise to blinding retinal dystrophies. The structures of the rod, especially those of the sensory cilium known as the outer segment, have been the subject of structural, biochemical, and genetic analysis for many years, but the molecular bases for rod morphogenesis and for cell death in rod dystrophies are still poorly understood. Recent developments in imaging technology, such as cryo-electron tomography and super-resolution fluorescence microscopy, in gene sequencing technology, and in gene editing technology are rapidly leading to new breakthroughs in our understanding of these questions. A summary is presented of our current understanding of selected aspects of these questions, highlighting areas of uncertainty and contention as well as recent discoveries that provide new insights. Examples of structural data from emerging imaging technologies are presented.
Neurons that form ribbon-style synapses are specialized for continuous exocytosis. To this end, their synaptic terminals contain numerous synaptic vesicles, some of which are ribbon associated, that have difference susceptibilities for undergoing Ca-dependent exocytosis. In this study, we probed the relationship between previously defined vesicle populations and determined their fusion competency with respect to SNARE complex formation. We found that both the rapidly releasing vesicle pool and the releasable vesicle pool of the retinal bipolar cell are situated at the ribbon-style active zones, where they functionally interact. A peptide inhibitor of SNARE complex formation failed to block exocytosis from either pool, suggesting that these two vesicle pools have formed the SNARE complexes necessary for fusion. By contrast, a third, slower component of exocytosis was blocked by the peptide, as was the functional replenishment of vesicle pools, indicating that few vesicles outside of the ribbon-style active zones were initially fusion competent. In cone photoreceptors, similar to bipolar cells, fusion of the initial ribbon-associated synaptic vesicle cohort was not blocked by the SNARE complex-inhibiting peptide, whereas a later phase of exocytosis, attributable to the recruitment and subsequent fusion of vesicles newly arrived at the synaptic ribbons, was blocked. Together, our results support a model in which stimulus-evoked exocytosis in retinal ribbon synapses is SNARE-dependent; where vesicles higher up on the synaptic ribbon replenish the rapidly releasing vesicle pool; and at any given time, there are sufficient SNARE complexes to support the fusion of the entire ribbon-associated cohort of vesicles. An important implication of these results is that ribbon-associated vesicles can form intervesicular SNARE complexes, providing mechanistic insight into compound fusion at ribbon-style synapses.
The connecting cilium of the rod photoreceptor is a tubular structure that bridges two adjacent cellular compartments, the inner segment, the major site of biosynthesis and energy metabolism, and the outer segment, a highly specialized ciliary structure responsible for phototransduction. The connecting cilium allows for active processes of protein sorting and transport to occur between them. Mutations affecting the cargo, their transporters, and the structural components of the primary cilium and basal body lead to aberrant trafficking and photoreceptor cell death. Understanding the overall design of the cilium, its architectural organization, and the function of varied protein complexes within the structural hierarchy of the cilium requires techniques for visualizing their native three-dimensional structures at high magnification. Here we describe methods for isolating retinas from mice, purifying fragments of rod cells that include much of the inner segment and the rod photoreceptor cilia, vitrifying the cell fragments, and determining their structures by cryo-electron tomography.
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