Mechanisms of Golgi Vesiculation during Mitosis

描述(由申请人提供)：我们用有丝分裂停滞的正常大鼠肾(NRK)细胞制备的有丝分裂液重建了渗透的正常大鼠肾(NRK)细胞的中心周围高尔基体堆的碎裂。在这些条件下，高尔基体膜以小的管状网状分子的形式散布在细胞质中。在我们的渗透细胞中重组的高尔基膜的碎裂相当于哺乳动物细胞有丝分裂周期的前中期/中期阶段的高尔基膜的状态。高尔基体碎裂需要丝裂原激活的激酶(MEK1)和类似Polo的激酶(PIK)。我们已经确定RAFL是高尔基体碎裂过程中MEK1的激活剂。我们的目的是证明raf1-MEK1-ERK(MAP Kinase)-GRASP55(高尔基相关蛋白)在高尔基体碎裂过程中的作用。我们将检验我们的工作假设，即一个名为CGNAP(中心体高尔基蛋白激酶N相关蛋白)的450kD大蛋白是PIK的下游靶点。我们认为PLK-CGNAP介导的反应将高尔基体膜从中央周围切断。Raf-MEK-ERK-GRASP55介导的途径使高尔基体相互断开。这些反应的最终结果导致高尔基体堆叠从中心周区分离出来，并以较小碎片的形式相互分离。此外，我们还发现着丝点周围高尔基体的碎裂是细胞进入有丝分裂所必需的。我们提出，中央周围高尔基体膜在进入有丝分裂的准备过程中起着“传感器”的作用。将讨论中央周围高尔基体调节这一关键事件的机制。我们的整体努力将提供对高尔基体膜调节进入有丝分裂的过程以及高尔基体膜在哺乳动物细胞中为有丝分裂特定事件做准备的机制的理解。

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.