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

本实验室从培养的人大肠杆菌中分离出四种推定的MAP， 通过与纯化玉米共聚的玉米细胞 微管 这些蛋白质将在几个方面进行表征。 不同层次 抗体将针对每一个 蛋白质，并将用于免疫荧光研究， 确定每一个的组织和亚细胞定位。 他们 将进行生物化学和体外功能表征 测定，以确定它们对微管聚合的影响， 稳定性和形态，以及它们可能的酶促或 机械化学活动 分子间的相互作用 微管的MAP将在一系列的研究中进行研究。 实验旨在确定微管结合位点， 微管上的MAP和MAP结合位点。 最后， cDNA克隆将被分离和表征用于 这些实验将在体内测试MAP功能。 微管是细胞骨架的主要组成部分之一 在所有真核细胞中。 它们代表了 细胞中各种重要的运动功能，包括 在有丝分裂或减数分裂期间染色体的物理分离， 纤毛和鞭毛运动，肌肉收缩， 亚细胞颗粒的细胞内转运等。 微管的活动被认为是由 微管相关蛋白或MAP。 然而，在植物中， 这些蛋白质还没有被证实。 植物微管 比动物更难处理 微管，以及那些容易工作的技术和程序， 动物源性材料对植物根本不起作用。 这 一个实验室最近开发了分离纯 微管，这是迈向 植物中微管组织和功能问题. 分子水平。 这项拟议中的工作有可能将 对植物的理解有了重大突破 在分子水平上的细胞骨架。

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.