Figure 10. Influence of the different treatments on actin dynamics.

(A) A stimulus that induces an increase in the cytosolic Ca²⁺ hastens SNARE-mediated fusion of secretory vesicles with the plasma membrane, but it also promotes both disruption of the preexisting actin network (green rosary beads) and the formation of new actin filaments (yellow rosary beads). This actin dynamics appears to favor the expansion of the fusion pore, but also prevents the collapse of the vesicle in the plasma membrane. This mechanism allows the fast release of soluble catecholamines (see the purple spike at the right). (B) The N-WASP inhibitor wiskostatin (Wks) strongly inhibits the new actin polymerization, while it slightly disturbs the preexisting cortical F-actin network (Figure 9). Such effect could be due to a reduction of the slow-rate of actin polymerization/depolymerization in resting conditions. Wsk also gives rise to incomplete release events (see the small green spike), suggesting that the lack of actin polymerization hinders the expansion of the fusion pore. Wsk could also perturb membrane transport, decreasing cellular ATP levels and Ca²⁺ entry [52]. These effects could also contribute to the failure of the fusion pore expansion. (C) Cytochalasin D (CytoD) greatly disrupts the preexisting F-actin network, as well as the new F-actin formation (Figure 7). CytoD also slows down the fusion pore expansion and increases the quantal size of the release events (see the big brown spike with a long foot). Probably the loss of actin meshwork favors the collapse of the vesicle in the plasma membrane, as previously proposed by Doreian et al. [8]. (D) The inhibition of Src kinases with PP2 suppresses the de novo F-actin formation, but it does not disturb the pre-existing cortical actin network (Fig. 2). PP2 also slows down the fusion pore expansion, and reduces the quantal size of the release events (note the small blue spike with a long foot). Similar effects were observed with the microinjections of cSrc-SH2 or -SH3 domains. These findings support the idea that the new F-actin formation favors the expansion of the fusion pore.