The proposed research has 3 separate but interrelated goals: 1) to clarify the role of the centrosome in organizing the cellular microtubule array, 2) to test the hypothesis that centrosomal separation is required for cellular progression from G1 into S phase in response to growth factors, and 3) to determine the physiological role of the endogenous colchicine like inhibitory protein (CLIP) in regulating microtubule assembly. 1) Monoclonal antibodies will be made to partially purified centrosomes and to HMW-MAPs. Those which react with the centrosome by immunofluorescence will be tested for their effects on microtubule regrowth from purified centrosomes in order to determine the role of the MAP related protein in microtubule nucleation and to identify other centrosomal proteins involved in this process. To define the contribution of the centrosome to cytoplasmic microtubule organization in vivo, anti-nucleating antibodies will be microinjected into cells under a variety of physiological conditions and their effect on the microtubule array studied by immunofluorescence. The effect of hormones and the cell cycle on biosynthesis and phosphorylation of nucleating proteins will be studied using immunoprecipitation. 2) Anti-centrosome monoclonal antibodies will be identified which, when microinjected into cells, inhibit centrosomal separation in response to growth factors and the effect of this inhibition on cellular entrance into S phase determined by autoradiography. The target proteins with which anti-separating antibodies react will be identified and the effect of growth factors on their biosynthesis and phosphorylation will be studied. Centrosomal calmodulin and actin binding proteins will be identified and studied to clarify their role in the mechanism of separation. 3) The binding of CLIP to intermediate filaments and the effects of this interaction on CLIP-tubulin binding will be studied in vitro. The distribution of CLIP in cells and its relationship to tubulin and intermediate filaments will be determined using immunofluorescence. To learn whether unpolymerized tubulin is associated with CLIP in living cells and to study the physiological significance of this interaction, anti-CLIP antibodies will be microinjected in order to disrupt or prevent tubulin-CLIP binding. The effects of CLIP inhibition on microtubule assembly and distribution will be defined by immunofluorescence. Hormonal and cell cycle regulation of CLIP synthesis and phosphorylation will be investigated using immunoprecipitation.
