Abstract
An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.
Highlights
Mast cells are specialized cells that respond to inflammatory signals by secreting large amounts of a wide range of inflammatory products
The cells were incubated in an extracellular solution containing blebbistatin (5 mM) for, 10 min before the pipette was sealed onto the plasma membrane
The upward and downward capacitance steps, which are associated with exocytosis and endocytosis, respectively, displayed slower kinetics in the cells treated with blebbistatin
Summary
Mast cells are specialized cells that respond to inflammatory signals by secreting large amounts of a wide range of inflammatory products. Exocytosis in mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and compound endocytosis, a mechanism by which the compound cavity, formed by the cumulative fusion of many secretory vesicles, is retrieved in a single membrane fission event [4]. In these modes of exo-endocytosis, the fused vesicles are not obliged to flatten, allowing the vesicles to be retrieved largely intact. These mechanisms do not require the effort of the invaginating membrane to form a deeply invaginated bud but do require the constriction and fission of the endocytic tubular neck to separate the vesicle from the plasma membrane and the inward movement of the vesicle into the cytosol [5,6]
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