Molecular and Biochemical Characterization of Microtubule- Associated Proteins from Zea mays

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This laboratory has isolated four putative MAPs from cultured cells of Zea mays (corn) by copolymerization with purified maize microtubules. These proteins will be characterized at several different levels. Antibodies will be raised against each of the proteins, and will be used in immunofluorescence studies to determine the tissue and subcellular localization of each. They will be characterized biochemically and in in vitro functional assays, to determine their effects on microtubule polymerization, stability, and morphology, and their possible enzymatic or mechanochemical activities. The molecular interactions of the MAPs with microtubules will be investigated in a series of experiments designed to determine microtubule binding sites on the MAPs and MAP binding sites on the microtubules. Finally, cDNA clones will be isolated and characterized for use in experiments that will test MAP function in vivo. Microtubules are one of the major kinds of cytoskeletal elements in all eukaryotic cells. They represent the structural basis for a wide variety of crucial motility functions in cells, including the physical separation of chromosomes during mitosis or meiosis, ciliary and flagellar movements, muscle contraction, intracellular transport of subcellular particles, etc. The activities of microtubules are presumed to be mediated by microtubule-associated proteins, or MAPs. However, in plants, no such proteins have yet been demonstrated. Plant microtubules have proven far more difficult to work with than animal microtubules, and techniques and procedures that work readily with animal-derived material do not work at all for plants. This laboratory has recently developed procedures for isolating pure microtubules, which was a necessary first step toward approaching the problem of microtubule organization and function in plants at the molecular level. The proposed work has the potential to turn into a major breakthrough in the understanding of plant cytoskeleton at the molecular level.