title: Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.
abstract: Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.
authors.affiliation: Genentech, South San Francisco, CA 94080, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Westlake
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Baye
authors.forename: Maxence V
authors.initials: MV
authors.lastname: Nachury
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Wright
authors.forename: Karen E
authors.initials: KE
authors.lastname: Ervin
authors.forename: Lilian
authors.initials: L
authors.lastname: Phu
authors.forename: Cecile
authors.initials: C
authors.lastname: Chalouni
authors.forename: John S
authors.initials: JS
authors.lastname: Beck
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.forename: Diane C
authors.initials: DC
authors.lastname: Slusarski
authors.forename: Val C
authors.initials: VC
authors.lastname: Sheffield
authors.forename: Richard H
authors.initials: RH
authors.lastname: Scheller
authors.forename: Peter K
authors.initials: PK
authors.lastname: Jackson
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D020788:Bardet-Biedl Syndrome / Q000378:metabolism / Q000503:physiopathology*; D002352:Carrier Proteins / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D002923:Cilia / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008566:Membranes / Q000254:growth & development; D015398:Signal Transduction / Q000502:physiology*; D059008:Time-Lapse Imaging; D014162:Transfection; D020798:Two-Hybrid System Techniques; D015027:Zebrafish; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C082377:RAB8A protein, human