Mechanisms of Golgi Vesiculation during Mitosis

描述（由申请人提供）：我们通过从有丝分裂停滞的NRK细胞制备的有丝分裂胞质溶胶，在透化的正常大鼠肾（NRK）细胞中重建了中心粒周围高尔基体堆叠的碎片。在这些条件下，高尔基体膜以小管网状元件的形式分散在整个细胞质中。在我们的透化细胞中重构的高尔基体膜的碎片化相当于哺乳动物细胞中有丝分裂周期的前中期/中期阶段的高尔基体膜的状态。高尔基体片段化需要丝裂原活化激酶激酶（MEK 1）和波罗样激酶（PIk）。我们已经确定Rafl作为高尔基体片段化过程中MEK 1的激活剂。我们的目的是证明Rafl-MEK 1-ERK（MAP激酶）-GRASP 55（高尔基体相关蛋白）在高尔基体片段化过程中的作用。我们将测试我们的工作假设，即称为CGNAP（中心体-高尔基体-蛋白激酶N -相关蛋白）的大的450 kD蛋白是PIK的下游靶标。我们建议PLK-CGNAP介导的反应切断高尔基体膜从pericentriolar区域。 Raf-MEK-ERK-GRASP 55介导的途径使高尔基体堆叠彼此断开。这些反应的最终结果导致高尔基体堆叠从中心粒周围区域分离，并且以较小片段的形式彼此分离。此外，我们还发现，高尔基体的碎裂是细胞进入有丝分裂所必需的。我们已经提出，高尔基体膜作为一个“传感器”，准备进入有丝分裂。的机制，其中pericentriolar高尔基体调节这一关键事件将得到解决。我们的整体努力将提供一个了解的过程中，高尔基体膜调节进入有丝分裂和高尔基体膜破碎的机制，在哺乳动物细胞中的有丝分裂特异性事件的准备。

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.