Mechanisms of Golgi Vesiculation during Mitosis

有丝分裂期间高尔基体形成水泡的机制

• 批准号: 6582730
• 负责人: VIVEK MALHOTRA
• 金额: $ 31.92万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6582730
• 关键词: Golgi apparatus; HeLa cells; SDS polyacrylamide gel electrophoresis; cell cycle; enzyme activity; fluorescence microscopy; immunoprecipitation; membrane activity; membrane proteins; membrane transport proteins; mitogen activated protein kinase; phosphorylation; protein sequence; transfection; western blottings

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.

描述（由申请人提供）：我们通过从在有丝分裂中滞留的NRK细胞制备的有丝分裂细胞溶胶中重构了透化正常大鼠肾（NRK）细胞中果皮三环堆积的碎片。在这些条件下，高尔基膜以分散在整个细胞质中的小微管鼠元素的形式。在我们的透化细胞中重新组成的高尔基膜的碎片等效于哺乳动物细胞中有丝分裂循环的胶质/变料阶段的高尔基膜状态。高尔基体碎片需要有丝分裂激活的激酶激酶（MEK1）和Polo像激酶（PIK）。我们已经将RAFL确定为高尔基分裂过程中MEK1的激活剂。我们的目的是证明Rafl-Mek1-ERK（MAP激酶）-grasp55（Golgi相关蛋白）在高尔基片段化过程中的作用。我们将测试我们的工作假设，即450 KD蛋白称为CGNAP（用于中心体-Golgi-蛋白酶激酶N-相关蛋白）是PIK的下游靶标。我们提出，PLK-CGNAP介导的反应会从周围三元素区域切断高尔基体膜。 RAF-Mek-Erk-Grasp55介导的途径彼此断开高尔基堆积。这些反应的最终结果导致高尔基堆积与周围三环区的分离，彼此以较小的片段形式分开。此外，我们发现丁香三元素高尔基体的碎裂对于将细胞进入有丝分裂是必要的。我们提出，丁香三元素高尔基膜起到了进入有丝分裂的准备的“传感器”。将解决该关键事件的果皮高尔基体调节这一关键事件的机制。我们的整体努力将对高尔基膜调节有丝分裂的进入以及高尔基膜分裂以准备哺乳动物细胞中有丝分裂特定事件的机制提供了一种理解。