Regulation of Coordination of Molecular Motors

分子马达协调的调节

• 批准号: 6778178
• 负责人: Vladimir I Gelfand
• 金额: $ 37.15万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6778178
• 关键词: Xenopus; cell line; chromatophore; cyclic AMP; cytoplasm; intracellular transport; macrophage; melanosomes; microfilaments; microtubules; molecular cloning; myosins; oncoprotein p21; organelles; phosphorylation; protein binding; protein kinase; protein structure function; site directed mutagenesis; thin layer chromatography; transfection; transport proteins

DESCRIPTION (provided by applicant): The transport of organelles and macromolecular complexes through the cytoplasm is essential for the function of every eukaryotic cell. This process is performed by motor proteins that move their cargo along microtubules and actin filaments. The spatial and temporal control of motor-dependent transport is critical for cell division, organelle transport and positioning, and the movement of RNA and protein complexes within the cell. The goal of this proposal is to understand the molecular mechanisms, which regulate organelle transport and coordinate the work of molecular motors. Our previous work established a permanent cell line of pigment cells (melanophores) from the frog Xenopus laevis as a model system to study the regulation of organelle movement. Movement of pigment organelles in these cells is initiated by changes in the concentration of cAMP and is performed by two microtubule motors of opposite polarity, cytoplasmic dynein and kinesin-II, and an actin motor, myosin-V. Organelle transport by these three motors is regulated by intracellular cAMP concentration. Furthermore, the activity of two microtubule motors is coordinated. This proposal has three specific aims: (i) find what regulates organelle transport along microtubules in the melanophore system: we will identify differentially regulated protein kinase(s) bound to organelles and determine how they regulate organelle transport; (ii) find the mechanism of coordination between plus and minus-end movement of pigment organelles along microtubules: we will determine if coordination is governed by cooperative differential binding of plus and minus-end directed motors to receptor proteins on the surface of organelles or it is determined by a stochastic mechanism; (iii) find how myosin-V is regulated in melanophores and other cell types: we will explore mechanisms of regulation of myosin-V in interphase cells using Xenopus melanophores and mouse macrophages and determine the role of p21-activated kinase (PAK1) in its regulation.

描述（由申请人提供）：细胞器和大分子复合物通过细胞质的运输对于每个真核细胞的功能至关重要。这个过程是由沿着微管和肌动蛋白丝移动其货物的运动蛋白执行的。运动依赖性运输的空间和时间控制对于细胞分裂、细胞器运输和定位以及细胞内 RNA 和蛋白质复合物的运动至关重要。该提案的目标是了解调节细胞器运输和协调分子马达工作的分子机制。我们之前的工作建立了来自非洲爪蟾的色素细胞（黑色素细胞）的永久细胞系，作为研究细胞器运动调节的模型系统。这些细胞中色素细胞器的运动由 cAMP 浓度的变化启动，并由两个相反极性的微管马达（细胞质动力蛋白和驱动蛋白-II）以及肌动蛋白马达（肌球蛋白-V）执行。这三个马达的细胞器运输受细胞内 cAMP 浓度的调节。此外，两个微管马达的活动是协调的。该提案有三个具体目标：（i）找到调节黑素细胞系统中微管细胞器运输的因素：我们将识别与细胞器结合的差异调节蛋白激酶，并确定它们如何调节细胞器运输； （ii）找到色素细胞器沿微管正负端运动之间的协调机制：我们将确定协调是否是由正负端定向马达与细胞器表面受体蛋白的协同差异结合控制的，还是由随机机制决定的； (iii) 发现肌球蛋白-V 在黑色素细胞和其他细胞类型中的调节机制：我们将利用非洲爪蟾黑色素细胞和小鼠巨噬细胞探索间期细胞中肌球蛋白-V 的调节机制，并确定 p21 激活激酶 (PAK1) 在其调节中的作用。