@article{fad7bfb104a64e869e3464c7f3f4db4f,
title = "Rab34 GTPase mediates ciliary membrane formation in the intracellular ciliogenesis pathway",
abstract = "The primary cilium is an essential organizing center for signal transduction, and ciliary defects cause congenital disorders known collectively as ciliopathies.1–3 Primary cilia form by two pathways that are employed in a cell-type- and tissue-specific manner: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium first forms inside the cell.4–8 After exposure to the external environment, cilia formed via the intracellular pathway may have distinct functional properties, as they often remain recessed within a ciliary pocket.9,10 However, the precise mechanism of intracellular ciliogenesis and its relatedness to extracellular ciliogenesis remain poorly understood. Here we show that Rab34, a poorly characterized GTPase recently linked to cilia,11–13 is a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by polarized MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and its paralog Rab36. Together, these findings establish that different modes of ciliogenesis have distinct molecular requirements and reveal Rab34 as a new GTPase mediator of ciliary membrane biogenesis.",
keywords = "GTPase, Rab, centriole, cilia, ciliary vesicle, ciliogenesis, ciliopathy, membrane",
author = "Ganga, {Anil Kumar} and Kennedy, {Margaret C.} and Oguchi, {Mai E.} and Shawn Gray and Oliver, {Kendall E.} and Knight, {Tracy A.} and {De La Cruz}, {Enrique M.} and Yuta Homma and Mitsunori Fukuda and Breslow, {David K.}",
note = "Funding Information: We acknowledge David Mick and members of the D.K.B. lab for advice and discussion, Derek Toomre (Yale University) for the In/Out reporter construct and cell line, Jadranka Loncarek (National Cancer Institute) for advice on expansion microscopy, Chris Westlake (National Cancer Institute) for cDNA encoding EHD1, Tamara Caspary (Emory University) for ARL13B cDNA, Tim Stearns (Stanford University) for Centrin2 cDNA, and Yong Xiong (Yale University) for pGro7 plasmid. This work was supported by funding from the National Institutes of Health ( R00HD082280 and R35GM137956 to D.K.B.; R35GM136656 to E.M.D.L.C.), the Charles H. Hood Foundation (D.K.B.), the Alfred P. Sloan Foundation ( FG-2018-10333 to D.K.B.), a Yale Anderson Endowed Fellowship (A.K.G.), the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Grant-in-Aid for Young Scientists 20K15739 to Y.H.; Grant-in-Aid for Scientific Research(B) 19H03220 to M.F.), the Japan Science and Technology Agency (CREST grant JPMJCR17H4 to M.F.), the Japan Society for the Promotion of Science (M.E.O.), and NSF major instrumentation grant 1725480 . S.G. was supported by R35GM136656-S1 . We acknowledge Felix Rivera-Molina, Derek Toomre, and the Yale CINEMA microscopy facility for assistance with SIM imaging; Yumei Wu, Xinran Liu, and the Yale Center for Cellular and Molecular Imaging for FIB-SEM sample processing and data collection; Ken Nelson and the MCDB FACS facility for assistance with cell sorting; Shirin Bahmanyar, Jing Yan, and Nadya Dimitrova for microscope use; Anna Pyle for phosphorimager use; Brandon Toyama for graphical assistance; and Kazuyasu Shoji for assistance with western blotting. Funding Information: We acknowledge David Mick and members of the D.K.B. lab for advice and discussion, Derek Toomre (Yale University) for the In/Out reporter construct and cell line, Jadranka Loncarek (National Cancer Institute) for advice on expansion microscopy, Chris Westlake (National Cancer Institute) for cDNA encoding EHD1, Tamara Caspary (Emory University) for ARL13B cDNA, Tim Stearns (Stanford University) for Centrin2 cDNA, and Yong Xiong (Yale University) for pGro7 plasmid. This work was supported by funding from the National Institutes of Health (R00HD082280 and R35GM137956 to D.K.B.; R35GM136656 to E.M.D.L.C.), the Charles H. Hood Foundation (D.K.B.), the Alfred P. Sloan Foundation (FG-2018-10333 to D.K.B.), a Yale Anderson Endowed Fellowship (A.K.G.), the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Grant-in-Aid for Young Scientists 20K15739 to Y.H.; Grant-in-Aid for Scientific Research(B) 19H03220 to M.F.), the Japan Science and Technology Agency (CREST grant JPMJCR17H4 to M.F.), the Japan Society for the Promotion of Science (M.E.O.), and NSF major instrumentation grant 1725480. S.G. was supported by R35GM136656-S1. We acknowledge Felix Rivera-Molina, Derek Toomre, and the Yale CINEMA microscopy facility for assistance with SIM imaging; Yumei Wu, Xinran Liu, and the Yale Center for Cellular and Molecular Imaging for FIB-SEM sample processing and data collection; Ken Nelson and the MCDB FACS facility for assistance with cell sorting; Shirin Bahmanyar, Jing Yan, and Nadya Dimitrova for microscope use; Anna Pyle for phosphorimager use; Brandon Toyama for graphical assistance; and Kazuyasu Shoji for assistance with western blotting. A.K.G. and M.C.K. designed and conducted most experiments and analyzed results. M.E.O. Y.H. and M.F. analyzed ciliogenesis in MDCK cells. K.E.O. analyzed Rab34 mutants, and T.A.K. conducted expansion microscopy. S.G. developed the recombinant protein purification protocol and performed biochemical analyses with purified protein components under the supervision of E.M.D.L.C. D.K.B. designed experiments, analyzed results, and supervised the project. D.K.B. and A.K.G. wrote the manuscript with input from all authors. The authors declare no competing financial interests. Publisher Copyright: {\textcopyright} 2021 Elsevier Inc.",
year = "2021",
month = jul,
day = "12",
doi = "10.1016/j.cub.2021.04.075",
language = "English",
volume = "31",
pages = "2895--2905.e7",
journal = "Current Biology",
issn = "0960-9822",
publisher = "Cell Press",
number = "13",
}