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性细胞器（包括线粒体、信号内体和病毒）在从轴突末端到细胞体的逆行方向上的基于微管的运输的马达。人类和小鼠中动力蛋白及其辅助蛋白的突变表明，适当的动力蛋白功能对神经生长、维持、再生和存活至关重要。在包括阿尔茨海默病和运动神经元病在内的广泛的神经退行性疾病中已经观察到基于动力蛋白的轴突运输的缺陷。动力蛋白是一种大型的多亚基蛋白复合物。中间链（IC）在复合物中起着关键作用，因为它与其他5个亚基和两个已知的动力蛋白活性调节因子相互作用。在脊椎动物中存在两种IC基因和IC的神经元特异性同种型。很少有人知道动力蛋白如何结合细胞器在轴突运输。虽然动力蛋白复合物被认为是最重要的结合蛋白，但有证据表明，动力蛋白与轴突中含有Trk的信号转导内体的结合还涉及另外的组分。我们发现，磷酸化的一个新发现的保守IC网站所需的动力蛋白结合到信号内涵体。这种磷酸化不调节动力蛋白与动力肌动蛋白的结合。我们将使用2D差异凝胶电泳来鉴定与磷酸化IC相互作用的Trk信号内体的组分，然后表征其在将动力蛋白募集到信号内体中的作用。虽然新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约束机制。我们将研究第二个IC磷酸化位点的作用，这可能对动力蛋白与内体的结合也很重要。动力蛋白被转运到轴突与细胞器在快速顺行运输。我们将使用生物化学和活细胞成像方法来识别和表征动力蛋白用于结合主要在顺行方向移动的细胞器的机制。这些研究将为动力蛋白与细胞器的结合提供一个新的模型，并为神经系统功能所必需的信号内体的轴突运输提供重要的见解。

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.