Molecular Mechanisms of Axonal Transport and Organelle Dynamics

项目摘要 分子马达驱动细胞器沿着细胞骨架的主动运输。这种运输是 在神经元中至关重要，高度极化的细胞将轴突延伸至1米。轴突不断地 由细胞体提供新合成的蛋白质和细胞器;主动清除老化蛋白质 并且功能失调的细胞器也是维持轴突稳态所必需的。因此，轴突运输由 细胞质动力蛋白和驱动蛋白马达的协调活动是必不可少的， 导致神经退化。在这里，我们集中在分子协调的动力蛋白和驱动蛋白马达在 通过支架蛋白和效应物的轴突运输，以及上游调节激酶和磷酸酶 在长的长度和时间尺度上保持持续的调节状态。我们也有兴趣 微管和肌动蛋白为基础的电机之间的相互作用，影响启动和终止 能动性最后，我们感兴趣的是分子马达和细胞骨架动力学的机制， 主动重塑细胞器膜，导致变形、变形、分裂和融合。我们将解决 这些问题使用活细胞成像和体外重建的协同方法， 分子分辨率来理解所涉及的机制。我们将重点实现三大目标。目标1： 了解细胞器运输的综合调节。每一种细胞器沿着沿着 轴突具有与其功能直接相关的独特的运动模式，但我们还没有完全理解轴突的运动模式。 调节这种运输的机制。我们将重点关注必要的轴突货物，自噬体和 信号内体，测试模型，货物特异性，整合调节电机允许 长时间和长距离的持续运输。在目标2中，我们寻求理解本地化的 在确定的轴突区域内调节细胞器动力学，包括轴突起始段， 突触前位点和轴突末端。这些区域呈现出独特的贩运模式， 细胞骨架组织的差异：微管束，加端动力学，翻译后 微管蛋白的修饰以及与肌动蛋白丝的交叉。我们感兴趣的机制， 运输启动、介导隔室特异性分选和控制货物的速率限制步骤 在细胞需要的特定部位递送/保留。在目标3中，我们将研究细胞器重塑驱动 通过相反的马达和/或细胞骨架动力学。当一些细胞器在细胞中移动时， 形态发生明显变化，其他货物发生急剧重塑，发生变形、裂变或 核聚变我们假设分子马达和细胞骨架丝提供了一个适应性工具箱， 被专门调整以调节动态细胞器形态。总之，这些方法应提供 轴突运输过程中细胞器动力学的重要新见解。由于轴突运输的缺陷导致 神经退行性疾病的进展可能为靶向和有效的治疗方法提供新的机会。

Project Summary Molecular motors drive the active transport of organelles along the cellular cytoskeleton. This transport is critically important in neurons, highly polarized cells that extend axons up to 1m. Axons are continuously supplied with newly synthesized proteins and organelles from the cell body; active clearance of aging proteins and dysfunctional organelles is also required to maintain axonal homeostasis. Thus, axonal transport driven by the coordinated activities of cytoplasmic dynein and kinesin motors is essential, and deficits in this transport cause neurodegeneration. Here we focus on the molecular coordination of dynein and kinesin motors during axonal transport by scaffolding proteins and effectors, and the upstream regulatory kinases and phosphatases that maintain a sustained regulatory state over long length- and time-scales. We are also interested in interactions between microtubule- and actin-based motors, which affect both the initiation and termination of motility. Finally, we are interested in the mechanisms by which molecular motors and cytoskeletal dynamics actively remodel organelle membranes, leading to deformation, tubulation, fission and fusion. We will tackle these questions using the synergistic approaches of live cell imaging and in vitro reconstitution with single molecule resolution to understand the mechanisms involved. We will focus on three major goals. Goal 1: Understanding the integrated regulation of organelle transport. Each type of organelle moving along the axon has a distinct pattern of motility that directly relates to its function, but we do not yet fully understand the mechanisms regulating this transport. We will focus on essential axonal cargos, autophagosomes and signaling endosomes, testing the model that the cargo-specific, integrated regulation of motors allows for sustained transport over long time scales and distances. In Goal 2, we seek to understand the localized regulation of organelle dynamics within defined axonal zones, including the axon initial segment, presynaptic sites, and the axon terminal. These zones exhibit distinct trafficking patterns that correspond to differences in cytoskeletal organization: microtubule bundling, plus-end dynamics, post-translation modifications of tubulin, and intersections with actin filaments. We are interested in mechanisms that enhance the rate-limiting step of transport initiation, mediate compartment-specific sorting, and control cargo delivery/retention at specific sites of cellular need. And in Goal 3, we will study organelle remodeling driven by opposing motors and/or cytoskeletal dynamics. While some organelles move through the cell with little evident change in morphology, other cargos are dramatically remodeled, undergoing tubulation, fission or fusion. We hypothesize that molecular motors and cytoskeletal filaments provide an adaptable toolbox that can be specifically tuned to regulate dynamic organelle morphology. Together, these approaches should provide important new insights into organelle dynamics during axonal transport. As deficits in axonal transport lead to neurodegeneration, progress may provide new opportunities for targeted and effective therapeutic approaches.