Regulation of retrograde cargo transport in axons

轴突逆行货物运输的调节

基本信息

项目摘要

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.
1)轴突中线粒体逆行运输的调节 轴突中最重要的细胞器之一是线粒体。线粒体执行许多对局部微环境重要的功能,包括:1)产生细胞代谢所需的能量; 2)缓冲钙离子水平;和3)供应ATP以用于调节神经兴奋性的离子转运蛋白的正常功能。此外,线粒体的位置已被证明可以调节轴突分支。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,线粒体还需要移动以维持自身的健康:线粒体经历分裂-融合动力学,允许蛋白质,脂质和线粒体DNA的交换。如果这些动力学被破坏,线粒体迅速发生降解。因此,线粒体运输对轴突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物调节线粒体转运的因素。 在我的博士后培训快结束的时候,我发现了一个突变体,它几乎没有所有的逆行线粒体运动。该细胞系中的致病突变导致Actr 10(肌动蛋白相关蛋白10)的缺失,Actr 10是动力蛋白相关复合物dynactin的已知组分。在体内分析actr 10突变体的线粒体运动显示缺乏逆行线粒体运动,但正常的顺行(非动力蛋白相关)运输。其他货物运输检测,包括溶酶体和动力蛋白电机本身,没有改变actr 10突变体。为了确定Actr 10是否真的是连接线粒体与逆行马达所必需的,我们从actr 10突变体和野生型同胞中进行了线粒体分馏实验。这些实验证实,Actr 10是动力蛋白马达与线粒体相互作用所必需的。然而,这种联系可能不是直接的,因为Actr 10没有已知的膜相关结构域。为了鉴定构成Actr 10和线粒体之间这种联系的蛋白质,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人,我们目前正在测试它们在轴突逆行线粒体运输中的作用。总之,我们的工作将定义的机制,动力蛋白线粒体附件的逆行运动,这个细胞器在轴突。 2)确定轴突中逆行货物运输的新调节剂 正向遗传学是一种理想的和公正的方式来确定蛋白质的关键功能在细胞过程中。我们已经在斑马鱼中启动了正向遗传筛选,以确定对轴突中特定货物的逆行运输重要的蛋白质。对于该筛选,我们使用转基因品系,其用细胞质GFP(绿色荧光蛋白)标记斑马鱼中的感觉和运动神经元轴突。由于未能进行逆行运输的货物随着时间的推移在轴突终末积累,我们可以使用GFP荧光指示剂筛选轴突终末大小的诱变家族,以鉴定逆行轴突运输中断的菌株。除了作为一个有效的筛选程序,在不同的发育阶段进行现场筛选的能力也为我们提供了必要的灵活性,以研究在同一动物中不同时间点发育的多种类型的轴突。此外,我们的转基因系含有第二个转基因,用红色荧光蛋白TagRFP标记线粒体。因此,我们的筛选也将使我们能够鉴定线粒体定位缺陷的突变株以及逆行转运中断的更一般的标志物。总之,这个屏幕将确定逆行货物运输和线粒体定位和轴突运动调节新的监管机构。这将推进我们定义轴突中货物特异性逆行运输机制的目标。

项目成果

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Catherine M Drerup其他文献

Catherine M Drerup的其他文献

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{{ truncateString('Catherine M Drerup', 18)}}的其他基金

Mechanism and function of retrograde mitochondrial transport in axons
轴突逆行线粒体转运的机制和功能
  • 批准号:
    10570955
  • 财政年份:
    2022
  • 资助金额:
    $ 58.39万
  • 项目类别:
Mechanism and function of retrograde mitochondrial transport in axons
轴突逆行线粒体转运的机制和功能
  • 批准号:
    10340724
  • 财政年份:
    2022
  • 资助金额:
    $ 58.39万
  • 项目类别:
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:
    8862557
  • 财政年份:
    2014
  • 资助金额:
    $ 58.39万
  • 项目类别:
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
  • 财政年份:
    2014
  • 资助金额:
    $ 58.39万
  • 项目类别:
In vivo analysis of the mechanisms of axon transport.
轴突运输机制的体内分析。
  • 批准号:
    8125867
  • 财政年份:
    2011
  • 资助金额:
    $ 58.39万
  • 项目类别:
Regulation of retrograde cargo transport in axons
轴突逆行货物运输的调节
  • 批准号:
    10007510
  • 财政年份:
  • 资助金额:
    $ 58.39万
  • 项目类别:
Regulation of retrograde cargo transport in axons
轴突逆行货物运输的调节
  • 批准号:
    10266546
  • 财政年份:
  • 资助金额:
    $ 58.39万
  • 项目类别:

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