Rab35 controls cilium length, function and membrane composition
8021-00425B) to JSA, by a postdoctoral fellowship from FCT (SFRH/BPD/32323/2006) to CS and by iNOVA4Health—UID/Multi/ 04462/2013, a program financially supported by FCT/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. This article is supported by the LYSOCIL project. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 811087. ; 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. ; publishersversion ; published