Abstract
Rab and Arl guanine nucleotide-binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G protein with endocytic recycling, actin remodelling and cytokinesis roles. Rab35 loss via siRNAs, morpholinos or knockout reduces cilium length in mammalian cells and the zebrafish left-right organiser (Kupffer's vesicle) and causes motile cilia-associated left-right asymmetry defects. Consistent with these observations, GFP-Rab35 localises to cilia, as do GEF (DENND1B) and GAP (TBC1D10A) Rab35 regulators, which also regulate ciliary length and Rab35 ciliary localisation. Mammalian Rab35 also controls the ciliary membrane levels of Shh signalling regulators, promoting ciliary targeting of Smoothened, limiting ciliary accumulation of Arl13b and the inositol polyphosphate 5-phosphatase (INPP5E). Rab35 additionally regulates ciliary PI(4,5)P2 levels and interacts with Arl13b. Together, our findings demonstrate roles for Rab35 in regulating cilium length, function and membrane composition and implicate Rab35 in pathways controlling the ciliary levels of Shh signal regulators.
Highlights
Primary cilia are microtubule-based organelles that protrude from the surface of most vertebrate cell types
In the proximal-most part of the cilium, the radial extent of the GFP-RAB35 signal is wider than that of ARL13B (Fig 1C and D, and Fig EV1A and B) and in ~20% of cells, GFP-RAB35 is more concentrated in this region (Fig EV1C). This proximal staining is reminiscent of the ciliary pocket localisation for the membrane remodelling Eps15 homology domain (EHD) 1 protein [76] and super-resolution imaging of GFP-RAB35 expressing hTERT-RPE1 cells stained for endogenous EHD1 revealed co-localization of both proteins in the proximal region of the cilium; importantly, these EHD1/GFP-RAB35 co-localising signals appeared broader than the radial extent of the ARL13B ciliary membrane signal, indicative of their association with the ciliary pocket membrane (Fig 1E)
We show evolutionarily conserved localisation of Rab35 at the ciliary membrane and demonstrate a role for Rab35 in restricting the ciliary accumulation of Arl13b and INPP5E and promoting the ciliary accumulation of SMO as well as PI(4,5)P2
Summary
Primary cilia are microtubule-based organelles that protrude from the surface of most vertebrate cell types. Operating as antenna-like structures, primary cilia detect and transmit chemical, light and mechanical signals from the extracellular environment to the intracellular space [1]. Primary cilia are critical for embryonic and postnatal development, serving key roles in important cell-cell communication signalling pathways (e.g. Sonic hedgehog, Wnt, PDGF) [2]. In the limb bud and developing nervous system, bone and neural tube patterning relies on the trafficking of Sonic hedgehog (Shh) signalling proteins into and out of cilia [3]. At the left-right organiser (LRO), or the equivalent Kupffer’s vesicle in zebrafish, the correct ratio of immotile and motile cilia [4] direct left-right patterning of the body plane via mechanisms that involve directional fluid flow, mechano- or chemo-sensation and planar cell polarity (PCP) signalling [5,6,7]. Defects in primary and motile cilia cause a wide range of mono- or multi-symptomatic ‘ciliopathy’ disorders such as primary ciliary dyskinesia, situs inversus, polycystic kidney disease, Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS) and Bardet-Biedl syndrome (BBS) which, collectively, affect most body tissues [8,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
