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

玉米微管相关蛋白的分子和生化特征

• 批准号: 8819304
• 负责人: Donald Fosket
• 金额: $ 4.16万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1989-09-01
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8819304&HistoricalAwards=false
• 关键词: Molecular; Biochemical; Characterization; Microtubule; Associated

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.

该实验室通过与纯化的玉米微管共聚，从玉米培养细胞中分离出了四种假定的 MAP。 这些蛋白质将在几个不同的水平上进行表征。 将产生针对每种蛋白质的抗体，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。 将对它们进行生化和体外功能测定，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化学活性。 MAP 与微管的分子相互作用将在一系列实验中进行研究，这些实验旨在确定 MAP 上的微管结合位点和微管上的 MAP 结合位点。 最后，cDNA 克隆将被分离和表征，用于测试体内 MAP 功能的实验。 微管是所有真核细胞中主要的细胞骨架元件之一。 它们代表了细胞中各种关键运动功能的结构基础，包括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运输等。微管的活动被认为是由微管相关蛋白（MAP）介导的。 然而，在植物中，尚未证实有这样的蛋白质。 事实证明，植物微管比动物微管更难使用，并且易于使用动物源性材料的技术和程序根本不适用于植物。 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中微管组织和功能问题的必要的第一步。 拟议的工作有可能成为分子水平上植物细胞骨架理解的重大突破。