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

动力蛋白转运复合物在动态微管上的组装和运动的时空分析

• 批准号: 313854929
• 负责人: Dr. Janina Baumbach
• 金额: --
• 依托单位: Europäisches Laboratorium für Molekularbiologie (EMBL)
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/313854929?language=en
• 关键词: Spatiotemporal; analysis; assembly; motility; dynein

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.

研究现状：细胞质动力蛋白是一种大的细胞骨架马达，能在细胞内主动运输细胞器、信使核糖核酸和病毒等多种物质。这一过程在延伸的神经细胞中尤其重要，动力蛋白运输缺陷会导致人类的神经疾病。动力蛋白行走的轨迹，即微管，是高度动态的结构，在生长和收缩之间交替。有证据表明，一组与生长中的微管正端(加上末端跟踪蛋白)特异结合的蛋白质对于将动力蛋白及其货物招募到微管轨道上是重要的。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分子的挑战和看似复杂的蛋白质相互作用网络，在细胞中很难研究动力蛋白在正端的募集。因此，我们对货物-动力蛋白复合体是如何组装的，以及运输是如何沿着动态微管启动和执行的仍然知之甚少。研究目的：本项目的目的是用纯化的蛋白质在体外重建动力蛋白在动态微管上的运输、起始和运动，从而剖析这一过程是如何被协调的。研究计划：我将提纯并荧光标记重组蛋白组件，以重建可运动的人动力蛋白复合体。我还将生产几种微管加末端跟踪蛋白的标记版本，这些蛋白与体内转运启动有关。我将使用这些蛋白质来重建沿着动态微管移动的动力蛋白，并使用能够分辨单分子的荧光显微镜技术来可视化这一过程。然后，我将加入不同的蛋白质，这些蛋白质结合到生长中的微管和末端，以确定从这些结构启动动力蛋白运输的最低限度的充分系统。接下来，我将通过拍摄标记有不同荧光团的蛋白质来剖析控制运输启动的事件序列，看看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在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体，并与之合作，使用负染色电子显微镜和冷冻电子显微镜研究它们的结构。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，以及它在神经疾病中是如何出错的。