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

本实验室通过与纯化的玉米微管共聚，从玉米(Zea Mays)培养细胞中分离到四个可能的图谱。这些蛋白质将在几个不同的水平上进行表征。将针对每种蛋白质产生抗体，并将用于免疫荧光研究，以确定每种蛋白质的组织和亚细胞定位。它们将在生化和体外功能分析中进行表征，以确定它们对微管聚合、稳定性和形态的影响，以及它们可能的酶或机械力化学活性。将在一系列实验中研究MAP与微管的分子相互作用，以确定MAP上的微管结合部位和微管上的MAP结合部位。最后，将分离和鉴定cdna克隆，用于在体内测试MAP功能的实验。微管是所有真核细胞中的主要细胞骨架成分之一。它们代表着细胞中一系列重要运动功能的结构基础，包括有丝分裂或减数分裂过程中染色体的物理分离、纤毛和鞭毛的运动、肌肉收缩、亚细胞内颗粒的运输等。微管的活动被认为是由微管相关蛋白或MAP介导的。然而，在植物中，还没有发现这样的蛋白质。事实证明，植物微管比动物微管更难处理，而容易处理动物来源材料的技术和程序对植物根本不起作用。该实验室最近开发了分离纯微管的程序，这是在分子水平上解决植物微管组织和功能问题的必要的第一步。这项拟议的工作有可能成为在分子水平上理解植物细胞骨架的重大突破。

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.