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: 6847190
• 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/6847190
• 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）：我们通过从有丝分裂停滞的NRK细胞制备的有丝分裂胞质，重建了透化的正常大鼠肾（NRK）细胞中中心粒周围高尔基体堆叠的碎片。在这些条件下，高尔基体膜以小管状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建的高尔基膜的碎片相当于哺乳动物细胞中有丝分裂周期的前中期/中期阶段的高尔基膜状态。高尔基体断裂需要丝裂原激活激酶 (MEK1) 和 Polo 样激酶 (PIk)。我们已经确定 Rafl 是高尔基体断裂过程中 MEK1 的激活剂。我们的目的是证明 Rafl-MEK1-ERK（MAP 激酶）-GRASP55（高尔基体相关蛋白）在高尔基体断裂过程中的作用。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 450 kD 蛋白是 PIK 的下游靶标。我们认为 PLK-CGNAP 介导的反应将高尔基膜与中心粒周围区域切断。 Raf-MEK-ERK-GRASP55 介导的通路使高尔基体堆栈彼此断开。这些反应的最终结果导致高尔基体堆叠与中心粒周围区域分离并以较小碎片的形式彼此分离。此外，我们发现中心粒周围高尔基体的破碎对于细胞进入有丝分裂是必要的。我们提出，中心粒周围高尔基体膜充当准备进入有丝分裂的“传感器”。将讨论中心粒周围高尔基体调节这一关键事件的机制。我们的总体努力将提供对高尔基膜调节进入有丝分裂的过程以及高尔基膜片段化为哺乳动物细胞中有丝分裂特异性事件做准备的机制的理解。