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

膜片钳电容测量可以直接分析活细胞质膜上单个融合和分裂事件的动力学和大小。这项技术为解开细胞外吞和内吞的机制提供了一个有吸引力的工具。因此，为了研究活的植物细胞内吞和外吞作用的潜在调节器的作用，我们将使用来自BY-2细胞培养的原生质体的电容测量作为模型系统。我们的研究重点将集中在当前囊泡融合和分裂的主题上，如细胞对渗透胁迫的反应，网状蛋白介导的内吞作用，以及肌醇磷脂参与外吞和内吞。应用特定的抑制剂和基因干扰内吞途径和磷脂酰肌醇的合成将被用来研究潜在的分子机制。此外，我们将建立利用花粉管进行膜片钳电容测量的方法，以提供一种新的工具来研究生理环境中的单个融合事件。这不仅将增加我们对花粉管分泌的了解，还将提供有关一般情况下胞吐的空间和时间调节机制的信息。

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.