Regulation of Coordination of Molecular Motors

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是必不可少的。这个过程是由马达蛋白完成的，马达蛋白沿着沿着微管和肌动蛋白丝移动它们的货物。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RNA和蛋白质复合物在细胞内的运动至关重要。该计划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。我们以前的工作建立了一个永久的色素细胞（黑色素细胞）的细胞系，从青蛙非洲爪蟾作为模型系统，研究细胞器的运动调节。这些细胞中的色素细胞器的运动是由cAMP浓度的变化引起的，并由两个极性相反的微管马达（细胞质动力蛋白和驱动蛋白-II）和一个肌动蛋白马达（肌球蛋白-V）进行。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）发现在黑色素细胞系统中是什么调节细胞器沿着微管的运输：我们将鉴定与细胞器结合的差异调节的蛋白激酶，并确定它们如何调节细胞器运输;（ii）发现色素细胞器沿着微管的正端和负端运动之间的协调机制：我们将确定是否协调是由合作的差异结合的正，负端定向马达受体蛋白的细胞器表面上或它是由一个随机机制决定;（iii）发现肌球蛋白-V在黑色素细胞和其他细胞类型中是如何调节的：我们将使用非洲爪蟾黑色素细胞和小鼠巨噬细胞探索肌球蛋白-V在间期细胞中的调节机制，并确定p21激活激酶（PAK 1）在其调节中的作用。

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.