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: Rab8 localizes to developing cilia but departs mature cilia. (A) Rab8 is an early marker of ciliogenesis. GFP-Rab8a localization in cilia was scored in live RPE GFP-Rab8a cells, and Actub-positive cilia were counted in fixed cells (n = 75 cells). (B) Rab8 accumulates in the growing cilia. One hour after serum withdrawal, RPE GFP-Rab8a cells transiently expressing tRFP-Centrin2 for 24 h were imaged every 10 min by SDC microscopy over 6 h with concurrent imaging in both red and green channels. Representative contrast-adjusted images shown are projections of 20 z-sections (step size 0.5 μm). (Bottom Row) 3D surface rendering. (Scale bar: 5 μm.) (Movie S1). (C) Kinetics of ciliogenesis. Keying on the initiation of nascent cilia, GFP-Rab8a levels in 12 developing cilia were measured from projected z-stacks collected as described in B. Regression analysis was performed on raw data (Inset) to determine the time required for GFP fluorescence to reach maximal levels (106 ± 12 min).

Article Snippet: GFP-Rab8a cilia localization was observed live by epifluorescence microscopy or Marianas spinning disk confocal (SDC) microscopy (Intelligent Imaging Innovations) at 40× magnification.

Techniques: Marker, Expressing, Microscopy, Imaging, Fluorescence

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: Serum withdrawal triggers Rabin8 association with vesicles that accumulate dynamically at the centrosome before ciliary membrane assembly. (A) Rabin8 localizes to dynamic centrosomal vesicles following serum withdrawal. Serum-starved (60 min) RPE GFP-Rabin8 cells were fixed and stained with the centriolar marker γ-tubulin (Movie S2). (Scale bar: 2 μm.) (B) Serum starvation causes transient association of Rabin8 with membrane vesicles and accumulation at the centrosome mediated by its COOH-terminal domain. Shown is quantification of GFP-Rabin8 and GFP-Rabin81–316 (ΔC)-positive vesicles and centrosomal accumulation in RPE cells (n = 50) (Fig. S2 A and B). (C) The Rabin8 COOH-terminal domain is required for ciliation. GFP-Rab8a–positive cilia were counted in cells transiently expressing control (tRFP), tRFP-Rabin8, and tRFP-Rabin81–316 for 72 h; serum was removed for the last 24 h. GFP-positive cilia were counted only in tRFP-positive cells (n = 50; P < 0.001). (D) Time course of RPE cells accumulating GFP-Rabin8 at the centrosome after serum removal. Fluorescence signal (black) has been inverted in D and E for better viewing of structures. (Scale bar: 2 μm.) n, nucleus. (E) Rabin8 centrosomal targeting precedes Rab8a ciliary membrane formation and is reduced after ciliogenesis. RPE GFP-Rabin8 cells transiently expressing tRFP-Rab8a for 24 h were imaged live every 30 min after serum withdrawal. GFP and tRFP images were collected separately. (Scale bar: 2 μm.)

Article Snippet: GFP-Rab8a cilia localization was observed live by epifluorescence microscopy or Marianas spinning disk confocal (SDC) microscopy (Intelligent Imaging Innovations) at 40× magnification.

Techniques: Staining, Marker, Expressing, Fluorescence