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细胞体和轴突末端之间的轴突运输对于轴突的生长、功能性突触的形成和神经元的存活至关重要。顺行运输（细胞体到轴突末端）依赖于大量的运动蛋白家族，而逆行货物运输（从轴突末端到细胞体）主要利用一种运动复合物，即细胞质动力蛋白。货物是如何选择性地与这个单一的马达结合并被运送到合适的位置的，这在很大程度上是未知的。据推测，这一过程涉及到连接蛋白，它将货物与核心动力蛋白运动复合物或其附属复合物动力蛋白结合。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，并确定这一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可及性，斑马鱼是研究完整脊椎动物逆行轴突运输及其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是高度保守的。为了开始解决我的目标，我使用前向遗传筛选来识别四种突变株，这些突变株显示的表型表明逆行货物运输中断，包括轴突末端肿胀。其中一株携带jnk相互作用蛋白3 （jp3）突变。初步分析显示，jip3突变体表现出长轴突截断和激活Ret （GDNF反应受体酪氨酸激酶）在突变轴突生长锥中的积累。在Aim 1中，我将讨论jp3作为Ret信号内体逆行运输所需的衔接蛋白的假设，这是轴突延伸所必需的。在我的筛选中发现的第二个突变体显示，由于细胞器逆行运输中断，轴突末端肿胀处线粒体积聚。线粒体的顺行运输和其他货物的逆行运输是正常的。该突变体的表型是由于Actr10的缺失，Actr10是动力蛋白附属复合体动力蛋白的一种已知成员。在Aim 2中，我将通过体内成像和Actr10蛋白相互作用域的生化解剖来确定Actr10是否作为介导线粒体逆行运输的适配器起作用。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额外新突变体中中断，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中有突变，所有这些突变在轴突运输中都具有未知的功能。最后，在Aim 4中，我将设计转基因斑马鱼菌株，用于鉴定Actr10相互作用组，并进一步解剖控制特定货物逆行轴突运输的分子机制。通过从实验中获得的数据、技能和工具，我将以独立调查员的身份破译各种货物逆行轴突运输的调制。

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.