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每个真核细胞的功能至关重要。这个过程是由运动蛋白完成的，它们沿着微管和肌动蛋白丝移动它们的货物。运动依赖转运的时空控制对细胞分裂、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。本研究的目的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一个永久的来自非洲爪蟾的色素细胞（黑色素细胞）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是由cAMP浓度的变化引发的，并由两个极性相反的微管马达（细胞质动力蛋白和动力蛋白ii）和肌动蛋白马达（肌动蛋白v）完成。这三个马达的细胞器运输受细胞内cAMP浓度的调节。此外，两个微管电机的活动是协调的。该提案有三个具体目标：(i)发现是什么调节了黑素细胞系统中沿微管的细胞器运输：我们将识别与细胞器结合的差异调节蛋白激酶(s)，并确定它们如何调节细胞器运输；（ii）发现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决定的，还是由随机机制决定的；（iii）发现myosin-V在黑色素细胞和其他细胞类型中的调节机制：我们将利用非洲蟾的黑色素细胞和小鼠巨噬细胞探索间期细胞中myosin-V的调节机制，并确定p21活化激酶（PAK1）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.