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First published online 9 May 2006
doi: 10.1242/jcs.02962

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Rab3A and Rab27A cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells

¹ Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
² Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan

View larger version (83K): [in a new window]   Fig. 1. Colocalization of Rab proteins with secretory vesicles in PC12 cells. NPY-Venus (A,D,G,J) and mRFP-Rab3A (B), mRFP-Rab27A (E), mRFP-Rab33A (H) or mRFP-Rab37 (K) were coexpressed in PC12 cells, and images of the fixed cells were obtained by confocal microscopy. Note that all four mRFP-Rabs colocalized well with the dense-core vesicle marker NPY-Venus. Bars, 5 µm.

View larger version (59K): [in a new window]   Fig. 2. Expression of Rab3A, Rab27A and Rab33A in PC12 cells. The same amount of recombinant FLAG-tagged Rab3A, Rab27A, Rab33A, Rab33B or Rab37 expressed in COS-7 cells (lanes 1-5) and total homogenates of PC12 cells (20 µg, lane 6) were loaded on 10% SDS-PAGE and immunoblotted with HRP-conjugated anti-FLAG (bottom panel), anti-Rab3A-, anti-Rab27A-, anti-Rab33A-, or anti-Rab37-specific antibody. Note that each of the four antibodies specifically recognized a single Rab isoform, and that Rab3A, Rab27A and Rab33A are endogenously expressed in PC12 cells. The positions of the molecular mass markers (x10-3) are shown on the left.

View larger version (67K): [in a new window]   Fig. 3. Silencing of endogenous Rab3A, Rab27A, and Rab33A with specific siRNAs. PC12 whole-cell lysates were prepared 2 days after transfection with Rab3A-siRNA (A), Rab27A-siRNA (C) or Rab33A-siRNA (E) as described in Materials and Methods. The knockdown effect of the siRNAs on endogenous expression of Rabs was evaluated by immunoblot analysis with specific antibodies (see also Fig. 2). The positions of the molecular mass markers (x10-3) are shown on the left. (B) PC12 cells transiently co-transfected with pEGFP-C1 and with a control vector (a-c) or with a vector containing Rab3A-siRNA (d-f). (D) PC12 cells transiently co-transfected with pEGFP-C1 and with a control vector (a-c) or with a vector containing Rab27A-siRNA (d-f). (F) PC12 cells transiently co-transfected with pEGFP-C1 and with a control vector (a-c) or with a vector containing Rab33A-siRNA (d-f). Note that expression of the specific siRNAs dramatically reduced endogenous expression of the Rab proteins (see arrowheads in panel e of B,D,F). Bars, 10 µm.

View larger version (53K): [in a new window]   Fig. 4. Effect of siRNA expression on the number of vesicles docked to the plasma membrane in PC12 cells. (A) Typical TIRF images of plasma membrane-docked vesicles before high-KCl stimulation of control (a), Rab3A-siRNA-expressing (b), Rab27A-siRNA-expressing (c) and Rab33A-siRNA-expressing cells (d). (e-h) Magnified images of the boxed area. Bars, 5 µm. (B) The density of docked vesicles was determined by counting the vesicles in each image (n=6 cells in each). The inset shows NPY-Venus and actin protein expression visualized with anti-GFP and anti-actin antibody, respectively. The positions of the molecular mass markers (x10-3) are shown on the left. (C) The number of NPY-Venus spot disappearance events was counted as fusion events in a 5-minute period (n=6 cells in each). The data are mean values ± s.e.m. and were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test. *P<0.05 and **P<0.01, respectively, compared with the control. Note that expression of either Rab3A or Rab27A siRNA significantly reduced the number of plasma membrane-docked vesicles (B) as well as the number of NPY-Venus release events (C).

View larger version (61K): [in a new window]   Fig. 5. Effect of the Rab3A-siRNA, Rab27A-siRNA, and Rab33A-siRNA expression on the kinetics of NPY-Venus release. (A) Typical sequential images of a single NPY-Venus vesicle observed after high-KCl (70 mM) stimulation of cells expressing control vector (control), Rab3A-siRNA (Rab3A siRNA), Rab27A-siRNA (Rab27A siRNA) or Rab33A-siRNA (Rab33A siRNA), acquired at 200-millisecond intervals, through a TIRF microscope. The third column of images (0.8 seconds) shows a diffuse cloud of NPY-Venus fluorescence, and the fourth column of images (1.2 second) shows disappearance of the spot. (B) Time course of the fluorescence changes measured in the center of NPY-Venus vesicles in the control (), Rab3A-siRNA-expressing (), Rab27A-siRNA-expressing (), and Rab33A-siRNA-expressing () cells. Mean fluorescence intensity before fusion was set as 100% (n=15 vesicles in each experiment). Bar, 2 µm. Images were acquired every 200 milliseconds under each set of conditions. Note that silencing Rab3A, Rab27A, or Rab33A had no effect on the kinetics of vesicle fusion of the dense-core vesicles.

View larger version (29K): [in a new window]   Fig. 6. Effect of simultaneous silencing of Rab3A and Rab27A on the number of vesicles docked to the plasma membrane in PC12 cells. (A) Confocal images of a PC12 cell showing colocalization of endogenous Rab3A (green signals in the left panel) and Rab27A (red signals in the middle panel). The right panel shows the overlay between Rab3A and Rab27A (yellow signals in the right panel). Scale bar=10 µm. (B) Typical TIRF images of plasma membrane-docked vesicles in a control cell (left) and a Rab3A- and Rab27A-siRNA-expressing cell (right). The bottom panels are magnified images of the boxed area. Scale bar=5 µm. (C) The density of docked vesicles was determined by counting the vesicles in each image (n=6 cells in each). (D) The number of NPY spot disappearance events in 5 minutes was counted as fusion events (n=6 cells in each). Data shown are mean values ± s.e.m. and were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test. *P<0.05, **P<0.01, and ***P<0.001, respectively, in comparison with the control. Note that simultaneous silencing of Rab3A and Rab27A caused a significant reduction of vesicle density than silencing of Rab3A or Rab27A alone.

View larger version (31K): [in a new window]   Fig. 7. Hypothetical model of dense-core vesicle docking mediated by Rab3A, Rab27A,and their effectors. At least seven Rab effectors (Rim1, Rim2, rabphilin, Noc2, Slp4-a, Slp5 and Slac2-c) are known to be expressed in endocrine cells. Three of them (rabphilin, Noc2 and Slp4-a) are capable of interacting with both Rab3A and Rab27A (Coppola et al., 2002; Fukuda et al., 2002; Haynes et al., 2001; Kotake et al., 1997; Kuroda et al., 2002a; Shirataki et al., 1993; Yi et al., 2002), whereas Rim interacts only with Rab3A (Fukuda, 2004; Fukuda, 2003a); Slac2-c and Slp5 specifically interact with Rab27A (Fukuda and Kuroda, 2002; Kuroda et al., 2002b). These Rab effectors simultaneously bind Rab27A on vesicle and plasma membrane proteins, plasma membrane-associated proteins or actin (see the text for details), and might be involved in the docking of dense-core vesicles to the plasma membrane (An and Almers, 2004; Betz et al., 2001; Cheviet et al., 2004a; Coppola et al., 2002; Desnos et al., 2003; Fukuda et al., 2003b; Fukuda et al., 2005; Imai et al., 2004; Torii et al., 2004; Tsuboi and Fukuda, 2005; Waselle et al., 2003).