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型的细胞骨架马达，在细胞内主动运输包括细胞器、mRNA和病毒在内的各种货物。这一过程在扩展的神经细胞中尤为重要，动力蛋白运输缺陷导致人类神经系统疾病。动力蛋白行走的轨迹，微管，是高度动态的结构，在生长和收缩之间交替进行。有证据表明，一组特异性结合生长的微管正端（正端跟踪蛋白）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。这一过程被认为是动态微管探测细胞空间并有效捕获货物的一种搜索和捕获机制。动力蛋白在正端的募集很难在细胞中进行研究，这是由于在体内观察单个分子和一个看似复杂的蛋白质相互作用网络的挑战。因此，我们对货物-动力蛋白复合物如何组装以及运输是如何沿着动态微管启动和执行的理解仍然很差。研究目的：本项目的目的是用纯化蛋白重建动力蛋白在体外动态微管上的运输起始和运动，从而剖析这一过程是如何被精心安排的。研究计划：我将纯化和荧光标记重组人体动力蛋白复合物所需的重组蛋白成分。我还将介绍几种微管和末端跟踪蛋白的标记版本，这些蛋白与体内运输起始有关。我将使用这些蛋白质沿着动态微管重建移动的动力蛋白，并使用能够分解单个分子的荧光显微镜技术将其可视化。然后，我将添加不同的蛋白质结合到生长的微管+端，以确定从这些结构开始运输动力蛋白的最小足够系统。接下来，我将通过拍摄标记有不同荧光团的蛋白质来分析控制运输起始的事件序列，以了解它们被招募到微管的顺序。最后，我将通过凝胶过滤层析纯化运输起始过程中形成的动力蛋白和正端跟踪蛋白的蛋白质复合物，并合作使用负染色电镜和冷冻电镜研究它们的结构。拟议的项目将使我们能够分析动力蛋白运输是如何在时间和空间上启动的，以及它在神经系统疾病中是如何出错的。

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.