Regulation of Coordination of Molecular Motors

描述(申请人提供)：细胞器和大分子复合体通过细胞质的运输对每个真核细胞的功能都是必不可少的。这个过程是由马达蛋白执行的，马达蛋白沿着微管和肌动蛋白细丝移动它们的货物。运动依赖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和蛋白质复合体在细胞内的运动至关重要。这项提议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非洲爪蛙色素细胞(黑素细胞)的永久细胞系，作为研究细胞器运动调节的模型系统。在这些细胞中，色素细胞器的运动是由cAMP浓度的变化启动的，并由两个相反极性的微管马达(细胞质动力蛋白和动力蛋白-II)和一个肌动蛋白马达(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。这个建议有三个具体的目的：(I)发现在黑素载体系统中是什么调节细胞器沿微管的运输：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的运输；(Ii)发现色素细胞器沿微管的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控制的，还是由随机机制决定的；(Iii)了解肌球蛋白V在黑素载体和其他细胞类型中的调控机制：我们将利用非洲爪哇黑素载体和小鼠巨噬细胞来探索肌球蛋白V在间期细胞中的调控机制，并确定p21激活激酶(PAK1)在其调控中的作用。

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.