Fig. 2.
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.)