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

Takashi Tsuboi 1 Mitsunori Fukuda 0 1 0 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 1 Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research) , 2-1 Hirosawa, Wako, Saitama 351-0198 , Japan - Recent studies have suggested that two small GTPases, Rab3A and Rab27A, play a key role in the late steps of dense-core vesicle exocytosis in endocrine cells; however, neither the precise mechanisms by which these two GTPases regulate dense-core vesicle exocytosis nor the functional relationship between them is clear. In this study, e we expressed a number of different Rab proteins, from c n Rab1 to Rab41 in PC12 cells and systematically screened ie them for those that are specifically localized on dense-core c S vesicles. We found that four Rabs (Rab3A, Rab27A, ll Rab33A, Rab37) are predominantly targeted to dense-core eC vesicles in PC12 cells, and that three of them (Rab3A, f Rab27A, Rab33A) are endogenously expressed on denselo core vesicles. We further investigated the effect of silencing a each Rab with specific small interfering RNA on vesicle n ru dynamics by total internal reflection fluorescence Jo microscopy in a single PC12 cell. Silencing either Rab3A Introduction The release of neurotransmitters and peptide hormones involves exocytotic fusion of secretory vesicles with the plasma membrane. Although the precise mechanism that allows the fine-tuning of exocytosis is still not well understood, a variety of exocytosis-regulating proteins have recently been identified, including SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), VAMP-2/synaptobrevin-2, SNAP-25 (synaptosome-associated protein of 25 kDa), syntaxin-1a and Rab GTPases (Jahn and Sdhof, 1999; 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 or Rab27A in PC12 cells significantly decreased the number of dense-core vesicles docked to the plasma membrane without altering the kinetics of individual exocytotic events, whereas silencing of Rab33A had no effect at all. Simultaneous silencing of Rab3A and Rab27A caused a significantly greater decrease in number of vesicles docked to the plasma membrane. Our findings indicate that Rab3A and Rab27A cooperatively regulate docking step(s) of dense-core vesicles to the plasma membrane. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/11/2196/DC1 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. Results Rab3A, Rab27A and Rab33A are endogenously ce expressed in PC12 cells and associated with dense-core n vesicles e ic To determine how many Rab isoforms specifically localize on S dense-core vesicles, we first performed a Rab-family-wide lle analysis by expressing our pool of Rab proteins (Rab1 to C Rab41), each tagged with green fluorescent protein (GFP) in fo PC12 cells. If certain Rabs were specifically targeted to densel core vesicles in NGF-differentiated PC12 cells, GFP-Rab a n proteins should only be present in the distal part of the neurites, ru the same as the endogenous Rab3A and Rab27A proteins Jo (Fukuda et al., 2002). Only three, phylogenetically similar, proteins of this Rab1 to Rab41 group (Fukuda, 2003a; PereiraLeal 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, o (...truncated)