Regulation of retrograde cargo transport in axons

1)轴突线粒体逆行转运的调控 轴突中最重要的细胞器之一是线粒体。线粒体对局部微环境具有许多重要的功能，包括：1)产生细胞新陈代谢所需的能量；2)缓冲钙离子水平；3)为调节神经兴奋性的离子转运体的正常功能提供ATP。此外，线粒体的位置已被证明调节轴突分支。线粒体不仅需要适当地定位在轴突中以维持轴突的健康和功能，而且为了维持自身的健康，线粒体也需要移动：线粒体经历了允许蛋白质、脂类和线粒体DNA交换的裂变-融合动力学。如果这些动力被打乱，线粒体就会迅速退化。因此，线粒体的运输对轴突的功能和健康至关重要。我们的实验室正在努力确定通过逆行马达蛋白复合体调节线粒体运输的因素。 在我的博士后培训接近尾声时，我发现了一个突变体，它几乎完全缺乏线粒体的逆行运动。该基因的致病突变导致Actr10(肌动蛋白相关蛋白10)的缺失，Actr10是动力蛋白相关复合体dynactin的已知成分。在体内对actr10突变体的线粒体运动的分析显示，线粒体缺乏逆行运动，但正常的顺行(与动力蛋白相关)运输。其他被检测的货物的运输，包括溶酶体和动力蛋白马达本身，在actr10突变体中没有改变。为了确定Actr10是否确实是将线粒体连接到逆行马达所必需的，我们对Actr10突变体和野生型兄弟姐妹进行了线粒体分离实验。这些实验证实，Actr10是动力蛋白马达与线粒体相互作用所必需的。然而，这种连接可能不是直接的，因为Actr10没有已知的膜相关结构域。为了确定Actr10和线粒体之间的这种联系的蛋白质，我们进行了免疫沉淀实验，然后进行了质谱分析。这些实验产生了一些有趣的候选者，我们目前正在测试它们在轴突中线粒体逆行运输中的作用。总之，我们的工作将确定动力蛋白-线粒体附着在轴突中这一细胞器逆行运动的机制。 2)发现轴突逆行运输的新调节因子 正向遗传学是识别在细胞过程中具有关键功能的蛋白质的一种理想和公正的方法。我们已经在斑马鱼中启动了正向遗传筛查，以确定对轴突中特定货物的逆行运输至关重要的蛋白质。在这次筛查中，我们使用了一种转基因品系，它用细胞质GFP(绿色荧光蛋白)标记斑马鱼的感觉和运动神经元轴突。由于无法进行逆行运输的货物会随着时间的推移在轴突终末积累，因此我们可以使用GFP荧光指示器筛选突变家族的轴突终末大小，以识别逆行轴突运输中断的菌株。除了是一种有效的筛查程序，在不同发育阶段进行活体筛查的能力也给了我们必要的灵活性，以研究同一动物在不同时间点发育的多种类型的轴突。此外，我们的转基因系包含第二个转基因，用红色荧光蛋白TagRFP标记线粒体。因此，我们的筛查也将使我们能够识别线粒体定位缺陷的突变株，以及更普遍的逆行转运中断的标志。总之，这一筛选将识别逆行货物运输的新调节器，以及轴突中线粒体定位和运动性的调节器。这将推进我们的目标，即确定货物在轴突中逆行运输的机制。

1) Regulation of retrograde mitochondrial transport in axons One of the most crucial organelles in axons are mitochondria. Mitochondria perform many functions important for local microenvironments including: 1) generate the energy necessary for cellular metabolism; 2) buffer calcium ion levels; and 3) supply ATP for the proper functioning of ion transporters that regulate neural excitability. In addition, the location of mitochondria has been shown to regulate axon branching. Not only do mitochondria need to be properly localized in axons to maintain axon health and function, mitochondria also need to move in order for them to maintain their own health: Mitochondria undergo fission-fusion dynamics which allow the exchange of proteins, lipids, and mitochondrial DNA. If these dynamics are disrupted, mitochondria rapidly undergo degradation. Consequently, mitochondrial transport is of the utmost importance for axon function and health. Our lab is working to identify the factors that regulate mitochondrial transport by the retrograde motor protein complex. Towards the end of my post-doctoral training, I discovered a mutant which lacks almost all retrograde mitochondrial movement. The causative mutation in this line results in depletion of Actr10 (actin related protein 10) a known component of the dynein-associated complex, dynactin. In vivo analyses of mitochondrial movement in actr10 mutants revealed a lack of retrograde mitochondrial movement but normal anterograde (non-dynein related) transport. Transport of other cargos assayed, including lysosomes and the dynein motor itself, was not altered in actr10 mutants. To determine if Actr10 was in fact necessary to link mitochondria to the retrograde motor, we performed mitochondrial fractionation experiments from actr10 mutants and wildtype siblings. These experiments confirmed that Actr10 is necessary for the dynein motor to interact with mitochondria. This linkage is likely not direct, however, as Actr10 does not have known membrane-associated domains. To identify the proteins which make up this link between Actr10 and mitochondria, we performed an immunoprecipitation experiment followed by mass spectrometry analysis. These experiments yielded a number of interesting candidates which we are currently testing for their role in retrograde mitochondrial transport in axons. Together, our work will define the mechanism of dynein-mitochondrial attachment for retrograde movement of this organelle in axons. 2) Identifying novel regulators of retrograde cargo transport in axons Forward genetics is an ideal and unbiased way to identify proteins with critical functions in cellular processes. We have initiated a forward genetic screen in zebrafish to identify proteins important for the retrograde transport of specific cargos in axons. For this screen, we are using a transgenic line that marks both the sensory and motor neuron axons in zebrafish with cytoplasmic GFP (Green Fluorescent Protein). Because cargos that fail to undergo retrograde transport accumulate over time in axon terminals, we can screen our mutagenized families for axon terminal size using the GFP fluorescent indicator to identify strains with disruptions in retrograde axonal transport. In addition to being an efficient screening procedure, the ability to screen live at various developmental stages also gives us the flexibility necessary to study multiple types of axons that develop at different time-points in the same animals. Additionally, our transgenic line contains a second transgene to label mitochondria with the red fluorescent protein TagRFP. Consequently, our screen will also allow us to identify mutant strains with defect in mitochondrial positioning as well as more general markers of retrograde transport disruption. Together, this screen will identify novel regulators of retrograde cargo transport and regulators of mitochondrial localization and motility in axons. This will advance our goal of defining the mechanisms of cargo-specific retrograde transport in axons.