Spatiotemporal analysis of the assembly and motility of dynein transport complexes ondynamic microtubules

研究现状：细胞质动力蛋白是一种大的细胞骨架马达，在细胞内积极运输各种货物，包括细胞器，mRNA和病毒。这一过程在扩展的神经元细胞中特别重要，动力蛋白的运输缺陷导致人类神经系统疾病。动力蛋白沿着行走的轨道，微管，是高度动态的结构，在生长和收缩之间交替。有证据表明，一组特异性结合到生长的微管加末端（加末端跟踪蛋白）的蛋白质对于将动力蛋白及其货物招募到微管轨道是重要的。这一过程被认为是一种搜索和捕获机制，通过这种机制，动态微管可以探测细胞空间并有效地捕获货物。由于在体内可视化单个分子和看似复杂的蛋白质相互作用网络的挑战，在细胞中难以研究动力蛋白向正末端的募集。因此，我们仍然有一个贫穷的了解如何货物动力蛋白复合物组装和运输是如何启动和执行沿着动态微管。研究目的：本研究的目的是利用纯化的蛋白质在体外重建动力蛋白在动态微管上的转运起始和运动，从而剖析这一过程是如何协调的。研究计划：我将纯化并荧光标记重组蛋白组分，以重建能动的人类动力蛋白复合物。我还将生产标记版本的几个微管加末端跟踪蛋白，已牵连在体内运输启动。我将用这些蛋白质来重构沿着沿着动态微管运动的动力蛋白，并使用能够分辨单个分子的荧光显微镜技术来可视化。然后，我将添加不同的蛋白质，结合到不断增长的微管加结束，以确定最低限度的足够系统的动力蛋白运输开始从这些结构。接下来，我将通过拍摄用不同荧光团标记的蛋白质来分析控制运输起始的事件顺序，以了解它们被招募到微管的顺序。最后，我将纯化蛋白质复合物的动力蛋白，加上末端跟踪蛋白，在运输过程中形成的凝胶过滤色谱法，并在合作，研究其结构，使用负染色电子显微镜和冷冻电子显微镜。该项目将使我们能够分析动力蛋白运输如何在时间和空间中启动，以及它如何在神经系统疾病中出错。

Current state of research: Cytoplasmic dynein is a large cytoskeletal motor that actively transports a variety of cargoes including organelles, mRNA and viruses inside cells. This process is particularly important in extended neuronal cells and defects in transport by dynein lead to neurological diseases in humans. The tracks that dynein walks along, microtubules, are highly dynamic structures that alternate between bouts of growth and shrinkage. There is evidence that a group of proteins that specifically bind to the growing microtubule plus end (plus end-tracking proteins) are important for recruiting dynein and its cargo to the microtubule track. This process has been suggested to serve as a search and capture mechanism by which dynamic microtubules probe the cellular space and efficiently capture cargoes. Recruitment of dynein to plus ends is difficult to study in cells due to the challenges of visualizing single molecules in vivo and a seemingly complex network of protein interactions. Thus, we still have a poor understanding of how cargo-dynein complexes are assembled and how transport is initiated and executed along dynamic microtubules. Research objective: The aim of this project is to reconstitute dynein transport initiation and movement on dynamic microtubules in vitro with purified proteins and thus dissect how this process is orchestrated. Research plan: I will purify and fluorescently label recombinant protein components required to reconstitute motile human dynein complexes. I will also produce labelled versions of several microtubule plus end-tracking proteins that have been implicated in transport initiation in vivo. I will use these proteins to reconstitute moving dynein along dynamic microtubules and visualize this using a fluorescent microscopy technique able to resolve single molecules. I will then add different proteins that bind to growing microtubule plus ends to identify the minimal sufficient system for dynein transport initiation from these structures. Next I will dissect the sequence of events that control transport initiation by filming proteins labelled with different fluorophores to see in which order they get recruited to the microtubule. Finally, I will purify protein complexes of dynein and plus end-tracking proteins that are formed during transport initiation by gelfiltration chromatography and, in collaboration, study their structure using negative stain electron microscopy and cryo-electron microscopy. The proposed project will allow us to analyse how dynein transport is initiated in time and space and how it goes awry in neurological diseases.