The Role of Cytoplasmic Dynein in Axonal Transport

描述(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。细胞质动力蛋白是以微管为基础的各种膜细胞器，包括线粒体、信号内体和病毒，从轴突末端逆行运输到细胞体的马达。在人类和小鼠中，动力蛋白及其附属蛋白的突变表明，适当的动力蛋白功能对于神经的生长、维护、再生和生存是必不可少的。在包括阿尔茨海默病和运动神经元疾病在内的多种神经退行性疾病中，已经观察到动力蛋白轴突运输的缺陷。动力蛋白是一个大的多亚单位蛋白质复合体。中间链(IC)在复合体中起着关键作用，因为它与其他5个亚基以及两个已知的动力蛋白活性调节因子相互作用。脊椎动物中存在两种IC基因及其神经元特异性异构体。关于动力蛋白在轴突运输过程中是如何与细胞器结合的，人们知之甚少。虽然dynactin复合体被认为是最重要的结合蛋白，但有证据表明，另一个成分也参与了dynein与Trk的结合，其中包含轴突中的信号内吞。我们发现，新发现的保守IC位点的磷酸化是动力蛋白与信号内体结合所必需的。这种磷酸化不调节dynein与dynactin的结合。我们将使用2D差异凝胶电泳来鉴定Trk信号内体与磷酸化IC相互作用的成分，然后表征它在招募dynein到信号内体中的作用。虽然新的IC位点上的磷酸化对于动力蛋白与信号内体的结合是重要的，但我们的数据表明，动力蛋白与线粒体或其他细胞器的结合并不是必需的。这 为动力蛋白与货物结合的另一种机制提供了证据。我们将确定和描述其他绑定机制。我们将研究第二个IC磷酸化位点的作用，它可能也是动力蛋白与内体结合的重要因素。随着细胞器的快速顺行运输，动力蛋白被运输到轴突中。我们将使用生化和活细胞成像方法来识别和描述动力蛋白与主要沿顺行方向移动的细胞器结合的机制。这些研究将导致动力蛋白与细胞器结合的新模型，并对神经系统功能所必需的信号内小体的轴突运输有重要的见解。

DESCRIPTION (provided by applicant): Neurons require axonal transport to establish and maintain axons and synapses. Cytoplasmic dynein is the motor for microtubule-based transport of various membranous organelles, including mitochondria, signaling endosomes, and viruses, in the retrograde direction from the axon terminus to the cell body. Mutations in dynein and its accessory proteins in humans and mice demonstrate that proper dynein function is essential for nerve growth, maintenance, regeneration, and survival. Defects in dynein based axonal transport have been observed in a wide range of neurodegenerative diseases including Alzheimer's disease and Motor Neuron Disease. Dynein is a large multi subunit protein complex. The intermediate chain (IC) plays a key role in the complex as it interacts with the other 5 subunits and with two known regulators of dynein activity. There are two IC genes in vertebrates and neuron specific isoforms of the IC. Little is known about how dynein binds to organelles during axonal transport. While dynactin complex is thought to be the most important binding protein there is evidence that an additional component is also involved in dynein binding to Trk containing signaling endosomes in axons. We found that phosphorylation of a newly identified conserved IC site is required for dynein binding to signaling endosomes. This phosphorylation does not modulate dynein binding to dynactin. We will use 2D difference gel electrophoresis to identify the component of Trk signaling endosome that interacts with the phospho-IC and then characterize its role in recruiting dynein to signaling endosomes. While the phosphorylation on the new IC site is important for dynein binding to signaling endosomes, our data indicate that it is not necessary for dynein binding to mitochondria or other organelles. This provides evidence for another mechanism for dynein binding to cargo. We will identify and characterize the other binding mechanisms. We will investigate the role of a second IC phosphorylation site that may also be important for dynein binding to endosomes. Dynein is transported into the axon in association with organelles moving in fast anterograde transport. We will use biochemical and live cell imaging approaches to identify and characterize the mechanism dynein uses to bind to organelles that primarily move in the anterograde direction. These studies will lead to a new model for dynein binding to organelles and important insights into the axonal transport of signaling endosomes which is essential for the function of the nervous system.