Mechanisms of Golgi Vesiculation during Mitosis

描述（由申请人提供）：我们利用在有丝分裂中停止的NRK细胞制备的有丝分裂细胞质，重建了渗透性正常大鼠肾（NRK）细胞中中心周围高尔基堆的碎片。在这些条件下，高尔基膜以分散在细胞质中的小管网状元件的形式存在。在我们的通透化细胞中重建的高尔基膜的断裂状态相当于哺乳动物细胞有丝分裂周期中期前/中期的高尔基膜状态。高尔基体断裂需要丝裂原活化激酶（MEK1）和波罗样激酶（PIk）。我们已经确定Rafl是高尔基断裂过程中MEK1的激活剂。我们的目的是证明Rafl-MEK1-ERK （MAP激酶）-GRASP55（一种高尔基体相关蛋白）在高尔基体断裂过程中的作用。我们将验证我们的工作假设，即一个名为CGNAP（中心体-高尔基蛋白激酶N -相关蛋白）的450 kD大蛋白是PIK的下游靶点。我们认为PLK-CGNAP介导的反应切断了高尔基体膜与中心周围区域的联系。Raf-MEK-ERK-GRASP55介导的通路将高尔基体相互断开。这些反应的最终结果导致高尔基堆从中心周围区域分离，并以较小碎片的形式彼此分离。此外，我们还发现，中心周围高尔基体的断裂是细胞进入有丝分裂所必需的。我们已经提出，中心周围高尔基膜作为一个“传感器”准备进入有丝分裂。中心周围高尔基体调节这一关键事件的机制将被讨论。我们的总体努力将提供对高尔基体膜调节进入有丝分裂的过程的理解，以及高尔基体膜在哺乳动物细胞有丝分裂特定事件的准备过程中断裂的机制。

DESCRIPTION (provided by applicant): We have reconstituted the fragmentation of the pericentriolar Golgi stacks in permeabilized Normal rat Kidney (NRK) cells by mitotic cytosol prepared from NRK cells that are arrested in mitosis. The Golgi membranes are found in the form of small tubulo-reticular elements dispersed throughout the cytoplasm under these conditions. The fragmentation of the Golgi membranes reconstituted in our permeabilized cells is equivalent to the state of the Golgi membranes in the pre-metaphase/metaphase stage of the mitotic cycle in mammalian cells. The Golgi fragmentation requires Mitogen Activated Kinase Kinase (MEK1) and Polo like Kinase (PIk). We have identified Rafl as the activator of MEK1 in the Golgi fragmentation process. Our aim is to demonstrate the role of Rafl-MEK1-ERK (MAP Kinase)-GRASP55 (A Golgi associated protein) in the Golgi fragmentation process. We will test our working hypothesis that a large 450 kD protein called CGNAP (for centrosome -Golgi -Protein Kinase N -Associated Protein) is the downstream target of PIK. We propose that PLK-CGNAP mediated reaction severs the Golgi membranes from the pericentriolar region. The Raf-MEK-ERK-GRASP55 mediated pathway disconnects Golgi stacks from each other. The net result of these reactions leads to the separation of the Golgi stacks from the pericentriolar region and each other in the form of smaller fragments. In addition we have found that the fragmentation of the pericentriolar Golgi apparatus is necessary for entry of cell into mitosis. We have proposed that the pericentriolar Golgi membranes act as a "sensor" in preparation for entry into mitosis. The mechanism by which the pericentriolar Golgi apparatus regulates this key event will be addressed. Our overall efforts will provide an understanding of the process by which Golgi membranes regulate entry into mitosis and the mechanism by which Golgi membranes are fragmented in preparation for mitosis specific events in the mammalian cells.