Cell Cycle Regulation of Microtubule Assembly

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9315700 Suprenant The proposed research is an investigation of how microtubule assembly and function is regulated during the cell cycle. A multidisciplinary approach will be used to test the hypothesis that a 77 kD echinoderm MT-associated protein (EMAP) is part of a multi-subunit G-protein complex whose function is regulated by phosphorylation and GTP- binding. Biochemical methods will be used to identify EMAP- binding proteins and direct photoaffinity labeling will determine whether any of these proteins are GTP-binding proteins. Immunological techniques will be used to characterize EMAP-dynamin interactions in vitro by immunoprecipitation and in vivo by immunogold labeling for electron microscopy. In vivo phosphorylation of EMAP and dynamin will be accomplished by 32-P labeling followed by immunoprecipitation. Microtubule assembly dynamics will be examined in immunodepleted interphase and mitotic extracts by video-enhanced differential contrast light microscopy. Affinity-purified anti-dynamin and anti-EMAP antibodies will be microinjected into sea urchin blastomeres to determine their effects on cell division. Genetically engineered EMAP constructs in baculovirus will be transfected into insect cells and examined for their effects on endogenous microtubule and organelle arrays. The completion of the proposed study will provide insight into the regulation of cellular morphogenesis during mitosis. %%% Microtubules are essential and dynamic components of the eukaryotic cytoskeleton, and are the mediators of such critically important cellular and intracellular motility phenomena as flagellar and ciliary beating (necessary for such phenomena as spermatozoan or protozoan swimming, movement of bronchial mucus, etc.), movement of particles and vesicles from one end of the cell to another (e.g., movement of synaptosomes containing neurotransmitters from the cell body of a nerve cell, where the synaptosomes are packaged, down to the tip of the axon, where t he contents are released by exocytosis) and separation of daughter chromosomes during cell division. The regulation of microtubules, in terms of their spatial organization, functional capabilities, and length and stability is not yet well understood, yet obviously is critical to the proper functioning of the cell. Certain proteins are known to be associated with microtubules, and are termed MAPs (microtubule-associated proteins); these are presumed to be involved in the regulation or mediation of microtubule- associated functions. Working with invertebrate cells as models, a novel MAP (termed EMAP) was found in sea urchins which, upon analysis, was shown to be similar to known regulatory subunits of G-proteins (a class of regulatory proteins in cells which are involved in a wide variety of signal transduction phenomena) and which was also shown to bind to dynamin, a GTP-ase implicated in certain microtubule- mediated vesicle movement phenomena. Based on these findings, a testable model has been developed in which dynamin and EMAP are postulated to be microtubule linker proteins that associate with each other, with microtubules, and with vesicles. These associations may in turn be regulated by GTP binding and phosphorylation. The experiments proposed in this project will test key parts of the model. The research promises to shed light on how the microtubule system works in cells. ***