Effect of Cytosolic Ca2+ and F-Actin Polymerization on Fast Endocytosis and Rapid Replenishment of Immediately Releasable Pool in Mouse Chromaffin Cells

CECILIA BORASSI; CARLOTA GONZÁLEZ-INCHAUSPE; FERNANDO D. MARENGO; MAURICIO MONTENEGRO; LUCIANA GALLO

Sheffield

Congreso; 19th International Symposium on Chromaffin Cell Biology; 2017

International Symposium on Chromaffin Cell Biology

The immediately releasable pool (IRP) of chromaffin cells is a group of ready releasable vesicles highly coupled to voltage dependent Ca2+ channels that is rapidly secreted in response to a brief stimulus. In our standard conditions (5 mM external Ca2+, 0.5 mM internal EGTA), the application of a 50 ms squared depolarization induces the exocitosis of 25±3 fF and a subsequent endocytosis which evolve exponentially with a time constant (τ) of 1.21±0.19 s. After that, IRP exocytosis recovers with a τ of 1.4±0.1 s. To study the effect of cytosolic calcium on endocytosis and vesicle replenishment we modified the cytosolic buffer capacity. The rise of internal BAPTA (0.5, 1, 2 and 4 mM) led to the acceleration of endocytosis kinetics and recovery, with time constants of 0.62±0.062 and 0.45±0.04 s respectively for 4 mM BAPTA. A possible explanation of this effect is the action of cytosolic Ca2+ on cortical actin polymerization, which promotes kiss and run over complete fusion. In fact, we observed a Ca+2-dependent cortical actin disassembly when permeabilized cells were incubated with variable free Ca2+ concentrations (0.01 to 10 µM). Moreover, from Fura-2 measurements we estimated that cells loaded with 4 mM of BAPTA presented approximately 0.01 µM free cytosolic Ca+2, which corresponds to a highly polymerized cortical actin. To test this hypothesis we induced cortical actin depolymerization by application of 2 uM cytochalasin D on cells preloaded with BAPTA 4 mM. This treatment caused significan cortical F-Actin depolimerization, and retarded endocytosis and recovery kinetics with time constants of 1.00±0.12 and 1.0±0.17 s respectively. Our results show a correlation between fast endocytosis and vesicular replenishment kinetics, which seem to be inhibited by cytosolic Ca2+. These effects could be in part mediated by the degree of cortical actin polymerization.