Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors

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.

The photoreceptor cilium and its diseases

“…This unexpected finding suggests either species differences between mouse and frog or indicates very high redundancy between Rabs. Our recent work in zebrafish supports a role for Rab8 in Rhodopsin trafficking: We confirmed the presence of Rab8 at the surface of OCVs using correlative light and electron microscopy and observed Rab8-positive particles moving along with Rhodopsin in the IS region with live imaging (Figure 2) [23].…”

“…and Syntaxin3 [23,26,27], explaining the observed massive accumulation of unfused vesicles in cc2d2a-/-zebrafish PRs.…”

An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

“…Rhodopsin uptake into Golgi-to-ciliadirected rhodopsin transport carriers (RTCs) is linked with the recruitment and activation of fusion regulators through the wellconserved ciliary trafficking network mediated by Arf4 binding the VxPx motif in rhodopsin (Ezratty et al, 2016;Wang and Deretic, 2014;Ward et al, 2011). The Arf4-based ciliary targeting complex is composed of, and is sequentially ordered by, the Arf GAP ASAP1 (Brown et al, 1998), a GAP and an effector of Arf4, and the Rab11-FIP3-Rabin8 dual effector complex, which ultimately activates Rab8, a regulator of carrier fusion with the periciliary plasma membrane (note that Rab8 and Rab11 both have a and b forms, Rabin8 is also known as RAB3IP, and FIP3 is also known as RAB11FIP3) (Bachmann-Gagescu et al, 2011;Deretic et al, 1995;Mazelova et al, 2009a;Moritz et al, 2001;Ojeda Naharros et al, 2017;Omori et al, 2008;Vetter et al, 2015a,b;Wang and Deretic, 2015;Wang et al, 2012).…”

Insights into photoreceptor ciliogenesis revealed by animal models

“…Rab8-coated vesicles accumulated at the apical inner segments suggesting CC2D2A is important for membrane protein trafficking in zebrafish (Bachmann-Gagescu et al, 2012; Mougou-Zerelli et al, 2009). Ojeda Naharros, et al, showed recently that CC2D2A plays a role in the docking of opsin-laden vesicles at the periciliary membrane (Ojeda Naharros et al, 2017). By contrast, the Cc2d2a −/− mouse recapitulates features of Meckel syndrome with multiorgan defects (Veleri et al, 2014).…”