Mechanisms underlying coordinated regulation of exo- and endocytosis in BY-2 cells

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Patch-clamp capacitance measurements allow the direct analysis of the kinetic and size of single fusion and fission events at the plasma membrane of living cells. This technique provides an attractive tool to unravel the mechanisms of exo- and endocytosis. To study the role of potential regulators of exo- and endocytosis in living plant cells we will therefore use capacitance measurements on protoplasts from BY-2 cell cultures as a model system. The focus of our investigations will be on current topics of vesicle fusion and fission such as the response of cells to osmotic stress, the role of clathrin mediated endocytosis and the involvement of phosphoinositides in exoand endocytosis. Application of specific inhibitors and genetic interference with the endocytotic pathway and phosphoinositide synthesis will be used to study the underlying molecular mechanisms. In addition, we will establish the use of pollen tubes for patch-clamp capacitance measurements to provide a new tool for the investigating of single fusion events in a physiological context. This will not only increase our knowledge of secretion in pollen tubes but also provide information about the mechanisms involved in spatial and temporal regulation of exocytosis in general.