Arl13b and the exocyst interact synergistically in ciliogenesis

Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is...

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Published in:	Molecular biology of the cell Vol. 27; no. 2; pp. 308 - 320
Main Authors:	Seixas, Cecília, Choi, Soo Young, Polgar, Noemi, Umberger, Nicole L, East, Michael P, Zuo, Xiaofeng, Moreiras, Hugo, Ghossoub, Rania, Benmerah, Alexandre, Kahn, Richard A, Fogelgren, Ben, Caspary, Tamara, Lipschutz, Joshua H, Barral, Duarte C
Format:	Journal Article
Language:	English
Published:	United States American Society for Cell Biology 15-01-2016 The American Society for Cell Biology
Subjects:	Abnormalities, Multiple ADP-Ribosylation Factors - genetics ADP-Ribosylation Factors - metabolism Animals Cerebellum - abnormalities Cilia - metabolism Eye Abnormalities Genetic Association Studies HeLa Cells Humans Kidney - metabolism Kidney Diseases, Cystic Life Sciences Mice Mice, Knockout Microtubules - metabolism Mutation NIH 3T3 Cells Retina - abnormalities Vesicular Transport Proteins - metabolism Zebrafish Zebrafish Proteins - genetics Zebrafish Proteins - metabolism
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Summary:	Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 PMCID: PMC4713133 These authors supervised the work equally. These authors contributed equally to this work. C.S. performed immunoprecipitations, affinity chromatography purification, the direct interaction assay, and the immunofluorescence in mammalian cell lines, under the supervision of D.C.B.; H.M. performed immunoprecipitations, under the supervision of D.C.B.; N.L.U. performed the mouse crosses, genotyping, and kidney dissections, under the supervision of T.C.; S.Y.C. performed the experiments using zebrafish, as well as the immunohistochemistry, immunofluorescence, and immunoblotting analysis of mouse kidneys together with X.Z., under the supervision of J.H.L.; M.P.E. designed experiments and performed the purification of Arl13b-GST under the supervision of R.A.K.; R.G. performed immunofluorescence in RPE1 cells, under the supervision of A.B.; P.N. performed the mouse crosses, genotyping, and kidney dissections of Sec10 conditional knockout mice, under the supervision of B.F.; the manuscript was written by C.S. and D.C.B.
ISSN:	1059-1524 1939-4586
DOI:	10.1091/mbc.E15-02-0061