Objectives: 

Through exo- and endocytosis, cells release and take up soluble material as well as adding and retrieving plasma membrane. Much information on exo-and endocytosis has been gained by studying single events in real time by electrophysiological techniques. The life of an endocytic vesicle begins from flat plasma membrane patches by a sequential process of invagination, bud formation and fission. The scission step requires the mechano-enzymatic function of dynamin, actin dynamics and possibly myosin motor proteins. Our aim is to study the possible role of nonmuscle myosin II in exo- and endocytosis.

Materials: 

We used membrane capacitance measurements in the configuration of perforated patch-clamp. Cells were treated with blebbistatin, a specific inhibitor that blocks ATP hydrolysis and thus motor activity.

Results: 

Cells treated with blebbistatin presented slower fusion kinetics of individual granules compared with controls as measured by analysis of capacitance steps. However, there was no clear difference in fission kinetics of individual endocytic granules in control and blebbistatin treated cells. Interestingly, fission pore conductance analysis showed that myosin II plays a major role in scission. After formation, the fission pore is constricted and lengthened before the final release of the vesicle into the cytoplasm. By treatment with blebbistatin, the fission pore is formed and constricted to a final size of about 2 nm, but lengthening and final scission are blocked or dramatically slowed.

Conclusions: 

These data showed blebbistatin affected fission by making vesicle closure incomplete and suggest a new role of nonmuscle myosin II in endocytosis.