Identifying modulators of dynein-based cargo motility

描述（由申请人提供）： 蛋白质和细胞器在神经元胞体和轴突终末之间的轴突运输对于轴突生长、功能性突触的形成和神经元存活是必需的。虽然顺行运输（细胞体到轴突末端）依赖于驱动蛋白马达蛋白的大家族，但逆行货物运输（从轴突末端到细胞体）主要利用一种马达复合物，即细胞质动力蛋白。货物是如何选择性地与这一马达结合并被运送到适当的位置的，这在很大程度上是未知的。据推测，这一过程涉及衔接蛋白，其将货物结合到核心动力蛋白马达复合物或其辅助复合物动力蛋白。我的长期目标是确定特定逆行货物运输的介质，定义它们的功能，并确定这个过程的中断如何影响电路的形成和活动。由于其独特的遗传工具和成像的可及性，斑马鱼是研究逆行轴突运输及其在完整脊椎动物中中断的功能后果的理想系统。重要的是，大多数调节轴突运输的细胞过程在哺乳动物和斑马鱼之间高度保守。为了开始实现我的目标，我使用正向遗传筛选来鉴定四种突变株，它们显示出指示逆行货物运输中断的表型，包括轴突终末轴突。其中一种菌株携带JNK相互作用蛋白3（Jip3）突变。初步分析表明，jip3突变体表现出截断长轴突和积累的激活Ret（GDNF反应性受体酪氨酸激酶）的突变轴突生长锥。在目标1中，我将阐述这样的假设：Jip 3作为Ret信号内体逆行运输所需的衔接蛋白，这对于轴突延伸是必要的。在我的屏幕上发现的第二个突变体显示，由于这个细胞器的逆行运输中断，线粒体在轴突终末鞘中积累。线粒体的顺行运输和其他物质的逆行运输正常。该突变体的表型是由于Actr10（动力蛋白辅助复合物dynactin的已知成员）的缺失所致。在目标2中，我将确定是否Actr10作为一个适配器介导的线粒体逆行运输的功能，使用在体内成像和生化解剖的相互作用域的Actr10蛋白。在目标3中，我将使用我建立的协议和新技术来确定特定货物的逆行运输是否在我的其他新突变体中被破坏，以及这些缺陷如何影响电路的功能。我的初步数据显示，这些菌株在已知的动力蛋白相互作用因子中有突变，所有这些都在轴突运输中具有未知的功能。最后，在目标4中，我将设计转基因斑马鱼品系，用于鉴定Actr10相互作用组，并进一步剖析控制特定货物逆行轴突运输的分子机制。凭借从拟议实验中获得的数据、技能和工具，我将作为一名独立研究者，准备破译各种货物的逆行轴突运输的调节。

DESCRIPTION (provided by applicant): Axonal transport of proteins and organelles between the neuronal cell body and axon terminals is essential for axon outgrowth, formation of functional synapses, and neuronal survival. While anterograde transport (cell body to axon terminal) relies on the large family of kinesin motor proteins, retrograde cargo transport (from axon terminals towards the cell body) primarily utilizes one motor complex, cytoplasmic dynein. How cargo binds to this single motor selectively and is transported to the proper location is largely unknown. It has been postulated that this process involves adaptor proteins, which bind cargo to either the core dynein motor complex or its accessory complex, dynactin. My long-term goals are to identify mediators of specific retrograde cargo transport, define their function and determine how disruption of this process impacts circuit formation and activity. Because of their unique genetic tools and imaging accessibility, zebrafish are the ideal system to study retrograde axonal transport and the functional consequences of its disruption in an intact vertebrate. Importantly, most cellular processes that regulate axonal transport are highly conserved between mammals and zebrafish. To begin addressing my goals, I used a forward genetic screen to identify four mutant strains that display phenotypes indicative of interrupted retrograde cargo transport, including axon terminal swellings. One of these strains carries a mutation in JNK-interacting protein 3 (Jip3). Preliminary analyses revealed that jip3 mutants exhibit truncation of long axons and accumulation of activated Ret (GDNF responsive receptor tyrosine kinase) in mutant axon growth cones. In Aim 1, I will address the hypothesis that Jip3 serves as an adaptor protein required for retrograde transport of Ret signaling endosomes, which is necessary for axon extension. The second mutant identified in my screen displays accumulation of mitochondria in axon terminal swellings due to interrupted retrograde transport of this organelle. Anterograde mitochondrial transport and retrograde transport of other cargos are normal. The phenotype in this mutant is due to loss of Actr10, a known member of the dynein accessory complex, dynactin. In Aim 2, I will determine whether Actr10 functions as an adaptor mediating retrograde transport of mitochondria using in vivo imaging and biochemical dissection of interaction domains in the Actr10 protein. In Aim 3, I will use my established protocols and new techniques to determine if retrograde transport of specific cargos is disrupted in my additional novel mutants and how these defects affect function of the circuit. My preliminary data show that these strains have mutations in known dynein interactors, all with unknown functions in axonal transport. Finally, in Aim 4, I will engineer transgenic zebrafish strains which will be used to identify the Actr10 interactome and further dissect the molecular mechanisms that govern retrograde axonal transport of specific cargos. With the data, skill sets, and tools acquired from the proposed experiments, I will be poised to decipher the modulation of retrograde axonal transport of various cargos as an independent investigator.