Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

doi: 10.1073/pnas.1018823108

Figure Lengend Snippet: The TRAPPII complex associates with the Rabin8 NH2-terminal domain and is required for Rabin8 centrosomal targeting and primary cilium assembly. (A) LAP-tag purification of Rabin8-associated proteins. (Upper) LAP-Rabin8 and LAP-Rabin81–142 were purified by tandem affinity from 293Trex cells, and the eluted proteins were analyzed by SDS/PAGE (4–12% gradient) and silver stained; then 14 equally spaced gel slices were cut. TRAPPC components had high-percentage peptide coverage in gel slices analyzed by LC-MS/MS corresponding to their predicted molecular weight (Table S1). (Lower) Rabin8-binding domains important for centrosomal vesicular trafficking. (B) TRAPPC9 colocalizes with GFP-Rabin8 on centrosomal vesicles after serum starvation. Micrographs show RPE GFP-Rabin8 cells grown with or without serum for 60 min and fixed and stained with anti-TRAPPC9 antibodies before confocal laser scanning microscopy. (Whole-cell images are shown in Fig. S4E.) (Scale bars: 2 μm.) (C) TRAPPC3, -C9, and -C10 are required for Rabin8 centrosome trafficking. RPE GFP-Rabin8 cells expressing tRFP-Centrin2 were treated with two different ablating TRAPPC protein siRNAs for 72 h (knockdown efficiency is shown in Table S2) followed by serum starvation for 1 h. GFP-Rabin8 subcellular localization was scored in live cells (n = 50) by comparing siRab11a+siRab11b-treated cells (score = 0; Fig. 3C) with siControl-treated cells (score = 5). Results from three independent experiments are shown (Fig. S4H). (D) TRAPPC3, -C9, and -C10 are required for ciliation. RPE cells were treated with siRNAs as in C, serum starved 24 h, and stained with Actub and pericentrin antibodies (n = 75 cells; P < 0.0001). Representative data from three independent experiments are shown.

Article Snippet: Antibodies used were TRAPPC9 (Proteintech), acetylated α-tubulin and γ-tubulin (Sigma), pericentrin (Novus), and GFP ( 3 ).

Techniques: Purification, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Molecular Weight, Binding Assay, Confocal Laser Scanning Microscopy, Expressing