Munc13-1 Translocates to the Plasma Membrane in a Doc2B- and Calcium-Dependent Manner

Abstract

Munc13-1 is a presynaptic protein activated by calcium, calmodulin, and diacylglycerols (DAG) that is known to enhance vesicle priming. Doc2B is another presynaptic protein that translocates to the plasma membrane (PM) upon elevation of internal calcium concentration ([Ca2+]i) to the submicromolar range, and increases both spontaneous and asynchronous release in a calcium-dependent manner. We speculated that Doc2B also recruits Munc13-1 to the PM since these two proteins have been shown to interact physiologically and this interaction is enhanced by Ca2+. However, this calcium-dependent co-translocation has never actually been shown. To examine this possibility, we expressed both proteins tagged to fluorescent proteins in PC12 cells and stimulated the cells to investigate the recruitment hypothesis using imaging techniques. We found that Munc13-1 does indeed translocate to the PM upon elevation in [Ca2+]i, but only when co-expressed with Doc2B. Interestingly, Munc13-1 co-translocates at a slower rate than Doc2B. Moreover, while Doc2B dislocates from the PM as soon as the [Ca2+]i returns to basal levels, Munc13-1 dislocates at a slower rate and a fraction of it accumulates on the PM. This accumulation is more pronounced under subsequent stimulations, suggesting that Munc13-1 accumulation builds up as some other factors accumulate at the PM. Munc13-1 co-translocation and accumulation was reduced when its mutant Munc13-1H567K, which is unable to bind DAG, was co-expressed with Doc2B, suggesting that Munc13-1 accumulation depends on DAG levels. These results suggest that Doc2B enables recruitment of Munc13-1 to the PM in a [Ca2+]i-dependent manner and offers another possible Munc13-1-regulatory mechanism that is both calcium- and Doc2B-dependent.

PI(4,5)P2-binding effector proteins for vesicle exocytosis

“…The C2 domains of Doc2b also exhibit Ca 2+ -dependent interactions with PI(4,5)P 2 [114] so Doc2b and Munc13-1 may be co-recruited to PI(4,5)P 2 domains as a complex. Munc13-1 dissociated more slowly than Doc2b from the membrane upon Ca 2+ level decreases consistent with a distinct mechanism for Munc13-1 stabilization at the membrane [111]. Munc13-1 is proposed to function at the membrane by promoting SNARE protein complex assembly by binding to syntaxin-1 [39, 102, 109, 110].…”

“…The translocation of Munc13-1 was very similar to the activation mechanism for PKC with initial Ca 2+ -dependent interactions with PI(4,5)P 2 mediated by the C2 domain and subsequent membrane stabilization by DAG interactions with the C1 domain [112]. Interestingly, Ashery and co-workers found that Ca 2+ -dependent Munc13-1 translocation was markedly enhanced by or dependent upon the overexpression of Doc2b [111], which interacts with Munc13-1 [113]. The C2 domains of Doc2b also exhibit Ca 2+ -dependent interactions with PI(4,5)P 2 [114] so Doc2b and Munc13-1 may be co-recruited to PI(4,5)P 2 domains as a complex.…”

“…Recent studies revealed that Ca 2+ influx promotes the translocation of Munc13-1 to the plasma membrane in PC12 cells [51, 111]. The translocation of Munc13-1-GFP into punctate domains on the plasma membrane was extremely rapid (<10s) in response to depolarization and Ca 2+ influx [51, 111].…”

“…Recent studies revealed that Ca 2+ influx promotes the translocation of Munc13-1 to the plasma membrane in PC12 cells [51, 111]. The translocation of Munc13-1-GFP into punctate domains on the plasma membrane was extremely rapid (<10s) in response to depolarization and Ca 2+ influx [51, 111]. Neutralization of the Ca 2+ -binding sites of the C2B domain was found to inhibit evoked vesicle exocytosis and to prevent the Ca 2+ -dependent translocation of Munc13-1, which indicated that PI(4,5)P 2 binding by C2B may mediate translocation [51].…”

The regulated secretory pathway in neuroendocrine cells

“…The group of Frédéric Meunier reviews recent insights of the role of the cortical acto-myosin network (3), whereas the role of different tethering and priming factors such as CAPS, Munc13, and Doc2 proteins is described by the groups of Ury Ashery and Tom Martin (4,5). Paanteha Moghadam and Meyer Jackson review how various synaptotagmins regulate fusion pore kinetics and control the mode of release (6).…”