Identifying modulators of dynein-based cargo motility

识别基于动力蛋白的货物运动调节剂

• 批准号: 8790875
• 负责人: Catherine M Drerup
• 金额: $ 9.07万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8790875
• 关键词: Active Biological Transport; Adaptor Signaling Protein; Address; Affect; Axon; Axonal Transport; Bacterial Artificial Chromosomes; Binding; Biochemical; Biochemical Genetics; Biological Assay; Calcium; Carrier Proteins; Cell physiology; Cells; Complex; Data; Defect; Deposition; Disease; Dissection; Dynein ATPase; Elements; Embryo; Endosomes; Engineering; Exhibits; Family; GDNF gene; Gene Transfer Techniques; Genetic; Genetic Screening; Goals; Growth Cones; Growth Factor; Homeostasis; Image; Imaging Device; Initiator Codon; Interruption; Kinesin; Larva; Light; Link; Location; MAPK8 gene; Maintenance; Mammalian Cell; Mammals; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Mitochondria; Molecular; Motor; Movement; Mutation; Neurodegenerative Disorders; Organelles; Phase; Phenotype; Positioning Attribute; Presynaptic Terminals; Process; Proteins; Protocols documentation; Reagent; Receptor Protein-Tyrosine Kinases; Recycling; Regulation; Research Personnel; Role; Sequence Analysis; Signal Transduction; Specificity; Swelling; Synapses; System; Techniques; Testing; Therapeutic; Transgenic Organisms; Work; Zebrafish; anterograde transport; axon growth; base; calcium metabolism; cell motility; deep sequencing; design; dynactin; in vivo; in vivo imaging; member; mutant; nervous system disorder; neural circuit; neuronal cell body; neuronal survival; novel; null mutation; promoter; public health relevance; research study; retrograde transport; skills; tool

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.

描述（由申请人提供）：神经元细胞体和轴突末端之间蛋白质和细胞器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至关重要。顺行运输（细胞体到轴突末端）依赖于驱动蛋白运动蛋白大家族，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体，即细胞质动力蛋白。货物如何选择性地与这台发动机结合并运输到正确的位置尚不清楚。据推测，这一过程涉及衔接蛋白，它将货物与核心动力蛋白运动复合物或其辅助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调解者，定义其功能并确定该过程的破坏如何影响回路的形成和活动。由于其独特的遗传工具和成像可访问性，斑马鱼是研究逆行轴突运输及其在完整脊椎动物中破坏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识别四种突变株，这些突变株显示出指示逆行货物运输中断的表型，包括轴突末端肿胀。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 突变体表现出长轴突的截短和突变体轴突生长锥中激活的 Ret（GDNF 反应性受体酪氨酸激酶）的积累。在目标 1 中，我将提出这样的假设：Jip3 是 Ret 信号内体逆行运输所需的衔接蛋白，而 Ret 信号内体是轴突延伸所必需的。在我的屏幕中发现的第二个突变体显示，由于该细胞器的逆行运输中断，线粒体在轴突末端肿胀中积累。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 的缺失所致，Actr10 是动力蛋白辅助复合物 dynactin 的已知成员。在目标 2 中，我将使用 Actr10 蛋白中相互作用域的体内成像和生化解剖来确定 Actr10 是否作为介导线粒体逆行运输的接头发挥作用。在目标 3 中，我将使用我已建立的方案和新技术来确定我的其他新型突变体中特定货物的逆行运输是否受到干扰，以及这些缺陷如何影响电路的功能。我的初步数据表明，这些菌株在已知的动力蛋白相互作用子中存在突变，所有这些突变在轴突运输中都具有未知的功能。最后，在目标 4 中，我将设计转基因斑马鱼品系，用于识别 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，我将准备以独立研究者的身份破译各种货物的逆行轴突运输的调节。