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First published online 9 May 2006
doi: 10.1242/jcs.02962
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Research Article |
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
* Author for correspondence (e-mail: nori{at}mail.tains.tohoku.ac.jp)
Accepted 21 February 2006
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Exocytosis, Rab3A, Rab27A, Small interfering RNA, Total internal reflection fluorescence microscopy
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Introduction |
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; Rothman, 1994). Rab proteins are monomeric GTPases of the Ras superfamily, which, together with their specific effector molecules, regulate multiple steps of vesicle transport, including vesicle motility, vesicle docking to specific compartments in cells and a membrane-fusion process (Pfeffer, 2001; Segev, 2001; Zerial and McBride, 2001). More than 60 distinct Rab proteins have been identified in mice and humans, and these proteins appear to regulate various types or steps of membrane trafficking (Zerial and McBride, 2001).
Recent evidence has indicated that Rab3A and Rab27A, two closely related Rab isoforms, are associated with secretory vesicles and involved in the regulation of exocytosis. First, Rab3A, Rab27A, and their effectors (i.e. Slp4-a/granuphilin-a, Slac2-c/MyRIP, Noc2, rabphilin and Rim
) are endogenously expressed in certain neuroendocrine cells (Cheviet et al., 2004a; Chung et al., 1995; Desnos et al., 2003; Fukuda et al., 2002; Fukuda et al., 2004; Regazzi et al., 1996; Waselle et al., 2003; Yi et al., 2002). Second, overexpression of Rab3A or Rab27A effectors (or their Rab-binding domain) modulates dense-core vesicle exocytosis in neuroendocrine cells (Cheviet et al., 2004a; Chung et al., 1995; Coppola et al., 2002; Desnos et al., 2003; Fukuda et al., 2002; Fukuda, 2003b; Fukuda, 2004; Fukuda et al., 2004; Sun et al., 2001; Waselle et al., 2003; Yi et al., 2002). As an example, two Rab27A effectors, Slp4-a and Slac2-c, are present on dense-core vesicles (Desnos et al., 2003; Fukuda et al., 2002; Waselle et al., 2003; Yi et al., 2002) and overexpression of Slp4-a in PC12 cells strongly inhibits dense-core vesicle exocytosis, whereas other members of the Slp family promote instead dense-core vesicle exocytosis (Fukuda et al., 2002; Fukuda, 2003b). However, it has never been elucidated whether any Rab proteins other than Rab3A and Rab27A are involved in the control of dense-core vesicle exocytosis, and whether such Rabs, or Rab3A and Rab27A themselves, function sequentially, redundantly, cooperatively or independently in dense-core vesicle exocytosis in neuroendocrine PC12 cells.
In this study, we screened for Rab members that are specifically localized on the dense-core vesicles in PC12 cells and found that Rab33A protein, in addition to Rab3A and Rab27A, is endogenously expressed on dense-core vesicles in PC12 cells. We further investigated the function of Rab3A, Rab27A and Rab33A in the motion of a single dense-core vesicle during exocytosis in PC12 cells by total internal reflection fluorescence (TIRF) microscopy, also called evanescent wave or evanescence microscopy (Axelrod, 1981), using vesicle-targeted fluorescent proteins (Tsuboi et al., 2000; Tsuboi et al., 2003; Tsuboi et al., 2004; Tsuboi et al., 2005; Tsuboi and Fukuda, 2005; Tsuboi and Rutter, 2003). Inhibition of Rab3A or Rab27A function by small interfering RNA (siRNA) substantially reduced the number of vesicles docked at the plasma membrane and decreased the number of single exocytotic events without affecting the kinetics of vesicle fusion. Simultaneous inhibition of Rab3A and Rab27A function by siRNA caused a further reduction in the number of vesicles docked at the plasma membrane as well as in the number of single exocytotic events. By contrast, no effect was observed in Rab33A-depleted PC12 cells. Cooperative roles of Rab3A and Rab27A in the docking step of dense-core vesicle exocytosis in PC12 cells are discussed based on our findings.
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Results |
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). Only three, phylogenetically similar, proteins of this Rab1 to Rab41 group (Fukuda, 2003a; Pereira-Leal et al., 2001), i.e. Rab3A, Rab27A and Rab37, were exclusively localized in the neurites of the NGF-differentiated PC12 cells; Rab33A was predominantly localized in the neurites but also in the Golgi-like structure (supplementary material Fig. 1A). By contrast, other Rabs were mainly present in the peri-nuclear regions, plasma membrane and/or cytoplasm, rather than in the neurites (summarized in supplementary material Fig. 1B).
To further determine whether the above four Rab proteins were actually present on dense-core vesicles, we simultaneously labeled the dense-core vesicle cargo and Rab proteins in PC12 cells by expressing mutant pH-insensitive yellow fluorescent protein (YFP)-tagged neuropeptide Y (NPY-Venus), which efficiently targets dense-core vesicles (Nagai et al., 2002) and also mRFP-Rab3A, mRFP-Rab27A, mRFP-Rab33A or mRFP-Rab37. Confocal microscopy showed that most NPY-Venus-positive vesicles (74.6±3.6%, n=5 cells) colocalized with mRFP-Rab3A-positive vesicles (Fig. 1A-C), confirming the efficient targeting of mRFP-Rab3A to NPY-positive vesicles (i.e. dense-core vesicles). Similarly, most mRFP-Rab27A-labeled (Fig. 1D-F; 87.6±2.6%, n=5 cells), mRFP-Rab33A-labeled (Fig. 1G-I, 94.5±4.6%, n=5 cells) and mRFP-Rab37-labeled (Fig. 1J-L, 79.3±5.2%, n=5 cells) structures colocalized with NPY-Venus. Endogenous expression of Rab3A, Rab27A, Rab33A and Rab37 in PC12 cells was finally determined by immunoblotting with specific antibodies (Fig. 2). Expression of Rab3A, Rab27A and Rab33A proteins in PC12 cells was readily detected, but no expression of Rab37 protein was observed under our experimental conditions (Fig. 2, lane 6).
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Silencing of Rab3A, Rab27A and Rab33A proteins with specific siRNAs
To investigate the role(s) of Rab3A, Rab27A and Rab33A proteins in PC12 cells, endogenous expression of Rab3A, Rab27A and Rab33A proteins was reduced with specific siRNAs against each Rab isoform (see Materials and Methods for details). The effect of the siRNAs on the number of plasma-membrane-docked dense-core vesicles and number of exocytotic responses was monitored by TIRF microscopy (Tsuboi et al., 2000; Tsuboi et al., 2004; Tsuboi et al., 2005; Tsuboi and Fukuda, 2005; Tsuboi and Rutter, 2003). Expression of the Rab3A, Rab27A and Rab33A siRNAs reduced endogenous expression of Rab3A (Fig. 3A, 85.7±4.7%, n=3), Rab27A (Fig. 3C, 72.4±5.6%, n=3) and Rab33A (Fig. 3E, 61.3±10.4%, n=3), respectively, in PC12 cells by 48 hours after transfection, but their expression levels were unaffected when cells were transfected with a pSilencer vector as a control (Fig. 3A,C,E). To verify at single-cell level the effect of these siRNAs on endogenous expression of their respective Rabs, we co-transfected the siRNAs together with a pEGFP-C1 vector as a transfection marker and examined them with an immunofluorescence microscope. Marked reductions of endogenous Rab3A, Rab27A and Rab33A signals by their respective siRNA were also observed in approximately 65% of the GFP-expressing PC12 cells (n>130 cells, six dishes per each experiment) (see Fig. 3B,D,F, arrowheads).
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Silencing of Rab3A or Rab27A by siRNA reduces the number of secretory vesicles docked at the plasma membrane and the number of exocytotic events
To explore the possible role of Rab3A, Rab27A and Rab33A in dense-core vesicle exocytosis (e.g. recruitment, docking and fusion), we silenced Rab proteins with siRNAs and monitored by TIRF microscopy the impact of silencing on the dynamics of single exocytotic events within 
100 nm beneath the plasma membrane of PC12 cells (Axelrod, 1981; Tsuboi et al., 2000; Tsuboi et al., 2003; Tsuboi et al., 2004; Tsuboi et al., 2005; Tsuboi and Fukuda, 2005; Tsuboi and Rutter, 2003). This was accomplished by labeling dense-core vesicles with NPY-Venus and counting the number of plasma membrane-associated vesicles in the presence of specific siRNAs by TIRF microscopy before stimulating the cells with high-KCl solution (70 mM). We first investigated the transfection efficiency of the siRNAs in NPY-Venus-expressing PC12 cells by immunofluorescence analysis. We found that 68.3±5.7%, 66.3±1.3% and 67.5±4.3% of NPY-Venus-expressing PC12 cells (n>125 cells, six dishes per experiment) showed a significant reduction of endogenous Rab3A, Rab27A and Rab33A signals, respectively (see also Fig. 3), indicating a high transfection efficiency when NPY-Venus and siRNAs were co-transfected. As shown in Fig. 4A, expression of either Rab3A or Rab27A siRNA, significantly reduced the number of plasma-membrane-docked vesicles (by 70.1% and 61.4%, respectively) (Fig. 4B), whereas expression of Rab33A siRNA had no effect. These effects are unlikely to be attributable to the differences in NPY-Venus expression levels in the Rab3A, Rab27A and Rab33A siRNA-expressing cells, because NPY-Venus expression levels did not differ much (inset in Fig. 4B).
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We next counted the total number of NPY-Venus release events in cells that express NPY-Venus together with either Rab3A, Rab27A or Rab33A siRNA during incubation with the high-KCl buffer. The number of NPY-Venus release events was reduced by 57.8% and 42.8% in Rab3A-siRNA-expressing and Rab27A-siRNA-expressing cells, respectively, compared with the control cells (Fig. 4C). Again, expression of the Rab33A-siRNA had no effect.
Effect of silencing Rab3A or Rab27A on vesicle fusion
To determine whether silencing of Rab3A or Rab27A inhibits the rate (or kinetics) of vesicle exocytosis, the dynamics of single-vesicle fusion events was analyzed in single NPY-Venus-expressing vesicles near the plasma membrane. Although exocytotic events were detected much less frequently in cells that expressed Rab3A or Rab27A siRNA compared with control cells or cells transfected with Rab33A siRNA (Fig. 4C), the kinetics of individual fusion events was identical in all cells (Fig. 5): stimulation with high-KCl buffer caused NPY-Venus-containing spots to brighten and expand suddenly during the release of the fluorescent peptide (Tsuboi et al., 2004), with an identical time course in all cells (Fig. 5B). Thus, these results strongly indicate that Rab3A and Rab27A regulate the docking of dense-core vesicles to the plasma membrane in PC12 cells, rather than modulate vesicle fusion (or the kinetics of vesicle fusion) itself.
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Discussion |
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; Kasai et al., 2005; Stinchcombe et al., 2001).
How do Rab3A and Rab27A cooperatively regulate the docking step of dense-core vesicle exocytosis? We think that the cooperative function of Rab3A and Rab27A might be explained by the diversity of Rab3A and Rab27A effectors expressed in endocrine cells (Cheviet et al., 2004b; Fukuda, 2005). At least seven Rab3A/27A effectors (Rim1/2, rabphilin, Noc2, Slp4-a, Slp5 and Slac2-c) were already shown to be expressed in certain endocrine cells (Fukuda, 2005). Interestingly, rabphilin and Noc2, previously characterized as Rab3A effectors (Haynes et al., 2001; Kotake et al., 1997; Shirataki et al., 1993), also function as Rab27A effectors (Cheviet et al., 2004a; Fukuda et al., 2004), and the Rab27A effector Slp4-a can interact with Rab3A (Coppola et al., 2002; Fukuda et al., 2002; Kuroda et al., 2002a; Yi et al., 2002). It has recently been proposed that Slp4-a promotes the docking of dense-core vesicles to the plasma membrane by simultaneously interacting with Rab27A on the vesicle and with syntaxin-1a/Munc18-1 on the plasma membrane (Coppola et al., 2002; Fukuda, 2003b; Fukuda et al., 2005; Torii et al., 2004), and that rabphilin controls the docking step of dense-core vesicle exocytosis by simultaneously interacting with Rab27A on the vesicle and with SNAP-25 on the plasma membrane (Fukuda, 2006; Tsuboi and Fukuda, 2005) (see Fig. 7). Further studies are needed to determine the exact function of Rab3A and/or Rab27A effectors in the dense-core vesicle docking step.
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), the lack of any effect of Rab37 expression on dense-core vesicle exocytosis may be explained by the notion that Rab37 effector(s) are not endogenously expressed in PC12 cells. It would be interesting to investigate the function of Rab37 in the docking process of histamine-containing granules in mast cells. Although Rab33A is endogenously expressed in PC12 cells, our results indicate that Rab33A is not involved in the late stages of dense-core vesicle exocytosis. Since Rab33A signals have also been found in Golgi-like structures (Zheng et al., 1998) in addition to dense-core vesicles, a possible function of Rab33A is to facilitate post-Golgi trafficking, which may promote delivery of immature secretory vesicles to the cell periphery. Alternatively, Rab33A might be involved in the control of endosome-to-Golgi transport and/or retrograde transport from late to early Golgi compartments (Zheng et al., 1998). Further studies are needed to determine the exact function of Rab33A in membrane trafficking in PC12 cells.
In summary, we have demonstrated that both Rab3A and Rab27A are present on the same dense-core vesicles in PC12 cells, and cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells. As far as we know, this is the first study to demonstrate a functional relationship between Rab3A and Rab27A in regulated exocytosis. Future studies on the roles of Rab3A and Rab27A effectors will clarify the mechanism by which the two exocytotic Rabs cooperatively regulate the docking step of dense-core vesicle exocytosis at the molecular level.
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Materials and Methods |
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; Imai et al., 2004).
Plasmid construction
The following pairs of nucleotides with a 19-base target site (bold) and a 9-base loop (italics) were used to generate siRNA-expression plasmids against rat Rab3A or Rab33A mRNA. Rab3A siRNA(+) primer (5'-GGACAACATTAATGTCAAGTTCAAGAGACTTGACATTAATGTTGTCCTTTTTT-3') and Rab3A siRNA(-) primer (5'-AATTAAAAAAGGACAACATTAATGTCAAGTCTCTTGAACTTGACATTAATGTTGTCCGGCC-3') for Rab3A siRNA; and Rab33A siRNA(+) primer (5'-ATCCCTCTTGTACCGTGATTTCAAGAGAATCACGGTACAAGAGGGATTTTTTT-3') and Rab33A siRNA (-) primer (5'-AATTAAAAAAATCCCTCTTGTACCGTGATTCTCTTGAAATCACGGTACAAGAAGGATGGCC-3') for Rab33A siRNA. The pairs of nucleotides were mixed, denatured at 94°C for 2 minutes, annealed at 72°C for 1 minute, gradually cooled to 4°C for 2 hours, and then subcloned into the ApaI and EcoRI sites of the pSilencer 1.0-U6 vector (Ambion, Austin, TX), which expresses short hairpin RNA under the control of the mouse U6 promoter. The plasmids obtained were referred to as pSilencer-Rab3A and pSilencer-Rab33A, respectively.
The Rab3A, Rab27A, Rab33A and Rab37 cDNA fragments (Fukuda, 2003a; Kuroda et al., 2002a) were subcloned into the BamHI/NotI site of the pmRFP-C1-gk vector (Tsuboi and Fukuda, 2005) modified from pmRFP-C1 (BD Clontech), by introducing a short Gly linker immediately downstream from mRFP (monomeric red fluorescent protein) (Campbell et al., 2002). Plasmid encoding NPY (neuropeptide Y)-Venus (pVenus-N1-NPY) was generously provided by Atsushi Miyawaki (Nagai et al., 2002). Other expression constructs (pEF-FLAG-Rab3A, pEF-FLAG-Rab27A, pEF-FLAG-Rab33A, pEF-FLAG-Rab33B, pEF-FLAG-Rab37, pSilencer-Rab27A and pEGFP-C1-Rabs) were prepared as described elsewhere (Fukuda, 2003a; Kuroda et al., 2002a; Kuroda and Fukuda, 2004; M.F., unpublished data).
Cell culture
PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 10% horse serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin, at 37°C under 5% CO2 (Fukuda et al., 2002). For differentiation of PC12 cells, the cells were treated with 100 ng/ml ß-nerve growth factor (NGF). COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin.
Immunoblotting
PC12 cells (one confluent 10-cm dish) were homogenized in buffer containing 1 ml of 50 mM HEPES-KOH pH 7.2, 150 mM NaCl, 0.5 mM GTP
S[guanosine 5'-O-(3-thiotriphosphate)] and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A) in a glass-Teflon Potter homogenizer by 10 strokes at 900-1000 rpm. After solubilization with 1% Triton X-100 at 4°C for 1 hour, the insoluble material was removed by centrifugation at 15,000 rpm for 10 minutes. The proteins were analyzed by 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting with anti-Rab3A mouse monoclonal antibody (1:250 dilution), anti-Rab27A mouse monoclonal antibody (1:1000 dilution), anti-Rab33A rabbit polyclonal antibody (1 µg/ml) and anti-Rab37 rabbit polyclonal antibody (4 µg/ml). Immunoreactive bands were visualized with HRP-conjugated goat anti-mouse IgG (1:10,000) or HRP-conjugated goat anti-rabbit IgG (1:10,000) and detected by enhanced chemiluminescence (ECL) (Amersham Biosciences, Buckinghamshire, UK).
pEF-FLAG-Rab vectors (4 µg of plasmids in total) were transfected into COS-7 cells (density of 7.5x105 cells per 10-cm dish the day before transfection) with Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Three days after transfection, the cells expressing FLAG-Rab3A, FLAG-Rab27A, FLAG-Rab33A or FLAG-Rab37 proteins were harvested and homogenized in 1 ml of the homogenization buffer described above. Proteins were analyzed by 10% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-FLAG tag (M2) antibody (1:10,000 dilution).
RNA interference
PC12 cells cultured on a 35-mm dish were co-transfected with 3 µg of pSilencer vectors and 1 µg of pEGFP-C1 vector as a marker of transfected cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Two days after transfection, cells were subjected to immunofluorescence analysis, and downregulation of endogenous Rab3A, Rab27A or Rab33A protein was confirmed visually. To evaluate the efficacy of the siRNA against each Rab, we counted the number of EGFP- (or NPY-Venus)-positive and endogenous Rab-negative cells and the total number of EGFP- (or NPY-Venus)-positive cells in three different observation fields of each culture dish, and then the transfection efficiency of the siRNAs was calculated. To quantitatively validate the knockdown effect of siRNA by immunoblotting, PC12 cells cultured on a 10-cm dish were co-transfected with 18 µg of pSilencer vectors and 6 µg of pEGFP-C1 vector. Two days after transfection, cell lysates were prepared as described above, and tested for expression of Rab3A, Rab27A and Rab33A with anti-Rab3A mouse monoclonal antibody (1:100 dilution), anti-Rab27A rabbit polyclonal antibody (2 µg/ml) and anti-Rab33A rabbit polyclonal antibody (2 µg/ml), respectively. Immunoreactive bands were visualized with HRP-conjugated goat anti-mouse IgG (1:10,000) or HRP-conjugated goat anti-rabbit IgG (1:10,000) and detected by ECL.
To assess whether silencing of Rab proteins affects dense-core vesicle biogenesis, we co-transfected PC12 cells (one confluent 10-cm dish) with pSilencer vectors (18 µg of plasmids in total) together with pNPY-Venus (6 µg of plasmids in total) with Lipofectamine 2000 according to the manufacturer's instructions. Three days after transfection, the cells expressing NPY-Venus together with either the Rab3A siRNA, Rab27A siRNA or Rab33A siRNA were harvested and homogenized in 1 ml of the homogenization buffer as described above. The proteins were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-GFP mouse monoclonal antibody (1:250 dilution) and HRP-conjugated goat anti-mouse IgG antibody (1:10,000 dilution).
TIRF microscopy
PC12 cells were cultured as described above (Fukuda et al., 2002). For total internal reflection fluorescence (TIRF) imaging, PC12 cells were plated onto poly-L-lysine-coated coverslips. Cells were co-transfected with 1 µg of pVenus-N1-NPY and either 3 µg of pSilencer (a vector control), pSilencer-Rab3A, pSilencer-Rab27A or pSilencer-Rab33A with Lipofectamine 2000 according to the manufacturer's instructions. For double knockdown of Rab3A and Rab27A, cells were triple-transfected with 1 µg of pVenus-N1-NPY, 3 µg of pSilencer-Rab3A and 3 µg of pSilencer-Rab27A. The imaging was performed in modified Ringer's buffer at 37°C (RB: 130 mM NaCl, 3 mM KCl, 5 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose, and 10 mM HEPES pH 7.4). Stimulation with high-KCl buffer was achieved by perfusion with RB containing 70 mM KCl (NaCl was reduced to maintain the osmolarity).
Exocytosis of NPY-Venus at the single-vesicle level was monitored with a TIRF microscope similar to that described previously (Tsuboi et al., 2000; Tsuboi and Fukuda, 2005; Tsuboi and Rutter, 2003). In brief, a high numerical aperture objective lens (Plan Apochromatic, 100x, NA=1.45, infinity corrected, Olympus, Tokyo, Japan) was mounted on an inverted microscope (IX81, Olympus). The incident light for total internal reflection illumination was introduced into the high numerical aperture objective lens through a single-mode optical fiber and two illumination lenses (IX2-RFAEVA-2, Olympus). To observe the NPY-Venus fluorescence image, we used a diode-pumped solid-state 488-nm laser (HPU50100, 20 mW, Furukawa Electronic, Chiba, Japan) for total internal fluorescence illumination and a band-pass filter (HQ535/30m, Chroma, Rockingham, VT) as an emission filter. The laser beam was passed through an electromagnetically driven shutter (VMM-D3J, Unibritz, Rochester, NY). The shutter was opened synchronously with an electron multiplier charge-coupled device camera (C9100-02, Hamamatsu Photonics, Hamamatsu, Japan), and the length of exposure was controlled by MetaMorph software (version 6.3, Universal Imaging Corporation, Downingtown, PA). Images were acquired every 400 milliseconds or as indicated. To analyze the TIRF imaging data, single exocytotic events were manually selected, and the average fluorescence intensity of individual vesicles in a 0.7 µm x 0.7 µm square placed over the center of the vesicle was calculated. To define a single docked vesicle, we processed each image by high-pass filter as described in Lang et al. (Lang et al., 2000) and then counted the number of plasma-membrane-docked vesicles at the entire cell surface in the evanescent field. The number of fusion events was manually counted for a 5-minute period. Data shown are the means ± s.e.m. of at least five individual experiments. Statistical differences between means were compared by one-way ANOVA followed by Newman-Keuls multiple comparison test with GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).
Confocal imaging
For microscopic analysis, PC12 cells were fixed with 4% paraformaldehyde (Wako Pure Chemicals, Osaka, Japan) for 20 minutes. For immunostaining, cells were permeabilized with 0.3% Triton X-100 for 2 minutes and blocked with blocking buffer (1% BSA and 0.1% Triton X-100 in PBS) for 1 hour. The cells were first immunostained with the primary antibodies and then with Alexa Fluor 488- and 568-labeled secondary IgG (1:5,000 dilution). Cells were examined for fluorescence with a confocal laser-scanning microscope (Fluoview 500, OLYMPUS), and the images of the cells were processed with MetaMorph software.
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Acknowledgments |
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Footnotes |
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), 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.
