Mechanisms of Golgi Vesiculation during Mitosis

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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.