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Am J Physiol Cell Physiol (June 10, 2009). doi:10.1152/ajpcell.00562.2008
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Submitted on November 4, 2008
Revised on May 6, 2009
Accepted on May 29, 2009

Dynasore Inhibits Rapid Endocytosis in Bovine Chromaffin Cells

Chia-Chang Tsai1, Chih-Lung Lin2, Tzu-Lun Wang2, Ai-Chuan Chou2, Min-Yi Chou2, Chia-Hsueh Lee2, I-Wei Peng2, Jia-Hong Liao2, Yit-Tsong Chen2, and Chien-Yuan Pan3*

1 Academia Sinica
2 National Taiwan University
3 National Taiwan Univeristy

* To whom correspondence should be addressed. E-mail: cypan{at}ntu.edu.tw.

Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole-cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 sec to a value lower than the pre-stimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time zero of the curve fitted to the decay phase were all about 110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis.







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