Abstract
Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane.
Highlights
Ciliopathies are an expanding group of human disorders caused by primary cilium dysfunction and unified by a wide array of overlapping phenotypes: cystic kidneys, central nervous system malformations and retinal degeneration among others [1,2,3]
Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals to the cells
Concentration and regulation of signal detection proteins in the ciliary membrane is tightly regulated through polarized vesicle trafficking and through a selective barrier at the ciliary base called the transition zone (TZ)
Summary
Ciliopathies are an expanding group of human disorders caused by primary cilium dysfunction and unified by a wide array of overlapping phenotypes: cystic kidneys, central nervous system malformations and retinal degeneration among others [1,2,3]. Primary cilia are involved in transduction of a variety of environmental signals to the cell, including morphogens and light. To serve this purpose, the ciliary membrane is enriched with specific receptors and channels required for signal detection [4]. Mutations in several genes encoding TZ proteins lead to Joubert syndrome (JBTS; OMIM: 213300), a representative ciliopathy characterized by a very specific cerebellar malformation–the molar tooth sign–and associated in 30% of cases with retinal involvement due to photoreceptor (PR) dysfunction [8]
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