Src Kinases Regulate De Novo Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells
Figure 2
Inhibition of Src activity reduces Ca2+-dependent actin polymerization.
(A–B) To analyze the impact of Src kinase inhibition on actin dynamics, ACCs were permeabilized in the presence of Alexa Fluor 488 G actin, 10 µM free Ca2+ and 10 µM of the Src inhibitor PP2 or its inactive analogue PP3. (C–D) To evaluate the drug effects on the organization of the cortical actin network, cells were permeabilized with digitonin for 6 minutes in the presence of PP2 (or PP3), then fixed and stained with 1 µM phalloidin-rhodamine B. (E–H) Intact cells were incubated for 20 min with 10 µM PP2 (or PP3), maintained in resting condition (E–F) or stimulated with 20 µM ionomycin for 10 s (G–H), then fixed and stained with 1 µM phalloidin-rhodamine B to confocal visualization. A, C, E and G show representative confocal images for each condition. Scale bar = 10 µm. B, D, F and H correspond to the quantification of the cortical actin fluorescence intensity, where data are means ± SEM from at least 16 cells per each condition (*p<0.05 compared with PP3). Note that Src kinases inhibition with PP2 reduces the new formation of cortical actin filaments, but it does not affect the cortical actin network in permeabilized or intact cells.