IFIBYNE   05513
INSTITUTO DE FISIOLOGIA, BIOLOGIA MOLECULAR Y NEUROCIENCIAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Membrane retrieval mechanisms operating in chromaffin cells after massive exocytosis
Autor/es:
ANDRéS PéREZ BAY; FERNANDO D. MARENGO
Lugar:
Pucón, Chile.
Reunión:
Simposio; 13th Internacional Symposium of Chromaffin Cell Biology; 2006
Institución organizadora:
Internacional Symposium of Chromaffin Cell Biology
Resumen:
After exocytosis, neurons and neuroendocrine cells have to retrieve the excess of plasmamembrane and refill the depleted pools of vesicles. To perform this task various endocytotic mechanisms seem to operate in these cells. This work tries to characterize in mice chromaffin cells the endocytotic and membrane cycling mechanisms activated in response to massive exocitosis induced by a variety of stimuli. To achieve this goal we used imaging techniques combined with fluorescent markers such as FM1-43 and dextrans. Depolarizations induced by 50mM K+ plus 2mM Ca2+ during 0.5-7 min show two kinetically discernible endocytotic components (Endo, Fig1): a rapid one appearing within 30 sec that overcompensates exocytosis, and a delayed one appearing 1.5-2.5 min after, resulting in complete compensation. Although endocytosis retrieves all the fused membrane, only a fraction was released again in a second round of exocytosis 30-50 min later (Exo2,Fig1). Interestingly, the evolution of the non releasable fraction (NRF,Fig1) was correlated with the delayed endocytotic component. Application of 3 min depolarization but in 0.5mM Ca2+ induced a smaller exocytosis (23±3%), and the resulting endocytosis was capable to recover most of the fused membrane in newly releasable vesicles (Exo2: 19±2%). Similarly, when more physiological stimuli were applied (50-200ìM nicotine or 0.2-1mM acetylcholine for 1-5 min) the exocytosis was between 11±4%-23±2%, and the retrieved membrane became totally releasable in less than 30 min. These results suggest that a strong supra-physiological stimulation, which recycles more than 50% of plasmamembrane, triggers an endocytotic mechanism contributing to maintain the cell surface but not providing new releasable vesicles in a short period. Localized internalization of fluorescent 40KD dextrans suggest that this mechanism might result in the formation of large membrane structures like cisternae or vacuoles. In agreement with this hypothesis, the effect of PI-3-P inhibitor LY-294002 which is known to block bulk endocytosis in bipolar cells (1), partially inhibited endocytosis and totally blocked the generation of the non releasable fraction. Finally the early endocytotic overcompensation observed at 30 sec was completely blocked by staurosporine and bisindolilmaleimide without interfering with Exo2, suggesting that previously described PKC dependent fast endocytosis (2) is not associated with fast recovery of secretory vesicles.