Vivek Malhotra - US grants

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.

We have identified a novel chemical ilimaquinone that vesiculates Golgi membranes and depolymerizes microtubules, while other intracellular organelles and cytoskeletal elements remain unaffected. Upon removal of IQ, both the microtubules and the Golgi complex rapidly reassemble to their original form. During this assembly process VGMs (for vesiculated Golgi membranes) first fuse to forms stacks of Golgi cisternae. This process does not require microtubules and can also take place at 16 degrees C but not at temperatures below 16 degrees C. The stacks then in the presence of microtubules and incubation at 37 degrees C congregate as a complex in the pericentriolar region. We have reconstituted the assembly of Golgi stacks from VGMs in semi-intact cells. This stack assembly has an obligate requirement for ATP, hydrolysis of GTP, incubation at 16-37 degrees C and cytosolic proteins. We have a unique opportunity to isolate the proteins that catalyze the fusion between VGMs and provide a structural scaffold for the step wise assembly of Golgi stacks. We want to characterize intermediates in the stack assembly process and define the minimum assembled structure that is functionally active in the vesicular protein transport. We will Isolate cytosolic GAFs (for Golgi Assembly Factors) required for the assembly of VGMs into stacks of cisternae. We want to isolate the mitotic counterparts of GAFs to test whether these proteins undergo any posttranslational modifications such as phosphorylation/dephosphorylation in association with cell-cycle events. The questions we want to address are whether these posttranslational modifications have a functional significance and what are the enzymes responsible for the regulation of these modifications during cell-cycle. Therefore, with a battery of purified GAFs, antibodies and cDNAs combined with a functional assay we want to elucidate the mechanism by which stacks of Golgi cisternae are maintained in interphase cells and built in each daughter cell after cell-division. This has never been attempted before, and therefore promises to reveal novel proteins involved in regulating the three dimensional organization of Golgi stacks and the significance of this organization in vesicular protein transport.

technology /technique development; animal tissue; plants; microscopy; lasers; human tissue; growth factor; carbohydrates; biomedical resource;

We have reconstituted the fragmentation and dispersal of Golgi membranes by incubation of permeabilized Normal Rat Kidney (NRK) cells with mitotic cytosol and an ATP regenerating system at 32 C. We find that mitotic cytosol depleted of p34cdc2 kinase (the kinase necessary for entry into mitosis) causes Golgi fragmentation. Depletion or inactivation of the cytosolic Mitogen activated protein kinase kinase (MEK1), however, inhibits Golgi fragmentation. Adding back MEK1 to MEK1 depleted mitotic cytosol restores Golgi fragmentation. We find that the downstream (and the relevant) cytosolic MAPkinase, ERK1 and ERK2 are not required for this process. Interestingly, a MAPkinase, recognized by an antibody with broad cross-reactivity to ERK2 is tightly associated with Golgi membranes. Our overall goals for this proposal are to understand the mechanism by which MEK1 is activated and test our hypothesis that Golgi MAPkinase is the immediate downstream target of activated MEK1. Our studies should reveal the identity of a regulator termed MEK1-kinase necessary for MEK1 activation, components that are involved in dispersal of the fragmented Golgi membranes and finally the Golgi membrane associated proteins, including the MEK1 substrate required Golgi fragmentation. These studies should provide an under- standing of the mechanism by which Golgi membranes undergo extensive fragmentation at the onset of mitosis.