Our aim is to understand the mechanism by which Golgi membranes of animal cells maintain their continuity during cell division. At the onset of mitosis in animal cells membranes of the Golgi complex breakdown into a large number of discrete fragments. These fragments are thought to distribute randomly between daughter cells, where they assemble to form a single copy of Golgi complex in each daughter cell. This breakdown/reformation process has been proposed to ensure that each daughter cell receives a copy of Golgi complex during cell division. The characteristics of Golgi-derived fragments, the mechanisms of their production, their distribution between daughter cells and their reassembly to form Golgi membranes however, remain elusive. Specifically, we plan to develop an in vitro assay to monitor the process of Golgi breakdown. For this interphase Golgi membranes, either isolated or in semi-intact cells, will be incubated with mitotic extracts from Xenopus eggs. The breakdown of Golgi membranes into discrete fragments will be monitored by following the distribution of Golgi specific markers by immunofluorescence. Our working hypothesis is that during this break down process two types of fragments are produced from each cisterna of the Golgi complex. Type one contains the cisterna specific resident proteins and type two contains polypeptides that play a structural role in the dynamics of Golgi cisternae. We will test this hypothesis by isolating mitotic Golgi fragments from the in vitro incubations by a combination of immunoaffinity adsorption and velocity and equilibrium sucrose density gradient centrifugation. We will prepare antibodies against the components of purified mitotic Golgi fragments. The mitotic events are thought to be controlled by posttranslational modifications that are initiated upon activation of MPF kinase. A number of polypeptides in mitotic Golgi may therefore, be present in a modified form compared to their native from in interphase Golgi. The collection of antibodies mentioned above will be used to identify such polypeptides and the respective modifications, to ascertain their function in the Golgi break down process.
NIH ExPorter
NIH ExPorter