The Role of Cytoplasmic Dynein in Axonal Transport

描述（由申请人提供）：神经元需要轴突运输来建立和维持轴突和突触。细胞质动力蛋白是各种膜细胞器（包括线粒体、核内体和病毒）从轴突末端逆行到细胞体的基于微管的快速运输的马达。正常的动力蛋白功能对神经生长、维持和再生至关重要，最近的研究结果表明，人和小鼠的动力蛋白复合物功能缺陷突变会导致运动神经元神经变性。动力蛋白是一种大型的多亚基蛋白复合物，目前对其在神经元中是如何调控以完成其多种功能的了解甚少。本提案将重点关注两个亚基的作用，这两个亚基是动力蛋白货物结合域的一部分，它们定义了动力蛋白、中间链和DYNLT （Tctex1和rp3）轻链的不同种群。神经元中有六种中间链的同工异构体，本研究的第一个目标集中在两种，一种是普遍存在的，足以在细胞中发挥管家功能，另一种中间链主要存在于神经元中。我们已经证明两者都存在于神经突、轴突和树突中。我们制备了一种稳定的神经细胞系，表达用GFP标记的普遍存在的同种异构体；证明它被纳入功能性动力蛋白复合物；并与膜结合的细胞器相结合。gfp动力蛋白的运动可以在培养神经元的活神经突和轴突中成像。细胞系和培养的神经元将用于测试动力蛋白调节的几种模型，通过使用活细胞成像结合RNAi和动力蛋白相互作用蛋白的显性负操作来分析动力蛋白的行为。我们发现在PC12细胞中，普遍存在的动力蛋白异构体参与神经营养因子受体激酶（Trk）信号内体运输，而在培养的海马神经元中，信号内体由神经元异构体运输。在本研究中，我们将以这些观察结果为基础，利用生化、分子和活细胞成像方法来确定信号内体与不同同种异构体动力蛋白特异性结合的机制。动力蛋白轻链也被认为直接介导动力蛋白与特定蛋白质的结合。虽然之前已经确定了几个DYNLT1 （Tctex1）结合伙伴，但我们发现纺锤体检查点蛋白Bub3是相关轻链DYNLT3 （rp3）的特异性结合伙伴。在第三个目标中，我们将研究DYNLT3的功能意义及其与有丝分裂的结合，并确定DYNLT3负责特定结合的不同结构域。我们还将研究这条轻链与丝蛋白a结合的功能意义。这些研究将为了解动力蛋白功能、信号内体的轴突运输以及轴突运输中断在神经退行性变中的作用提供重要的见解。公共卫生相关性：运动蛋白主动运输许多蛋白质和货物是神经元健康和正常功能所必需的。一种运动蛋白——动力蛋白的突变会导致运动神经变性。这个项目研究这个马达是如何附着在货物上的。

DESCRIPTION (provided by applicant): Neurons require axonal transport to establish and maintain axons and synapses. Cytoplasmic dynein is the motor for microtubule-based fast transport of various membranous organelles, including mitochondria, endosomes, and viruses in the retrograde direction from the axon terminus to the cell body. Proper dynein function is essential for nerve growth, maintenance and regeneration, as shown by the recent findings that human and mouse mutants defective in dynein complex function result in motor neuron neurodegeneration. Dynein is a large multi subunit protein complex and very little is known about how it is regulated to accomplish its diverse functions in neurons. This proposal will focus on the roles of two subunits that are part of the dynein cargo binding domain and which define distinct populations of dynein, the intermediate chains and the DYNLT (Tctex1 and rp3) light chains. There are six isoforms of the intermediate chains in neurons and the first aims of this proposal concentrate on two, one is ubiquitous and sufficient for housekeeping functions in cells, the other intermediate chain is found primarily in neurons. We have shown that both are present in neurites, axons, and dendrites. We have prepared a stable neural cell line expressing the ubiquitous isoform tagged with GFP; demonstrated that it is incorporated into functional dynein complexes; and associated with membrane bounded organelles. The movement of the GFP-dynein can be imaged in living neurites and axons of cultured neurons. The cell line and cultured neurons will be used to test several models for dynein regulation, by analyzing the behavior of the dynein using live cell imaging coupled with RNAi and dominant negative manipulation of dynein interacting proteins. We have found that in PC12 cells, the ubiquitous dynein isoform is involved in neurotrophin receptor kinase (Trk) signaling endosome transport, while in cultured hippocampal neurons signaling endosomes are transported by the neuronal isoform. In this proposal we will build on these observations and use biochemical, molecular, and live cell imaging methods to identify the mechanisms that underlies the specific binding of signaling endosomes to dyneins with the different isoforms. The dynein light chains are also thought to directly mediate dynein binding to specific proteins. While several DYNLT1 (Tctex1) binding partners had been previously been identified, we found that Bub3, a spindle checkpoint protein is a specific binding partner of the related light chain DYNLT3 (rp3). In the third aim we will investigate the functional significance of DYNLT3 and this binding to mitosis, and identify the distinct domains of DYNLT3 that are responsible for the specific binding. We will also examine the functional significance of the binding of this light chain to filamin A. These studies will provide important insight into dynein function, the axonal transport of signaling endosomes, and the role of disrupted axonal transport to neurodegeneration. PUBLIC HEALTH RELEVANCE: Active transport of many proteins and cargoes by motor proteins is required for the health and proper function of neurons. Mutations in one motor, dynein, result in motor neurodegeneration. This project investigates how this motor attaches to cargo.