Untersuchungen zu den molekularen Mechanismen der Interaktionen zwischen Endoplasmatischem Reticulum und Mikrotubuli unter Verwendung von Vaccinia Virus-Replikations-Komplexen als Modellsystem

虽然微管（MT）是已知的内质网（ER）和ER动力学的结构组织中发挥重要作用，相对知之甚少，这些相互作用的分子细节。目前的建议旨在使用一种新的体外系统，基于哺乳动物细胞，研究MT/ER相互作用的分子机制。它利用了观察到的牛痘病毒（vv）复制的细胞质位点完全被ER包裹，并且这些ER封闭的位点以MT依赖的方式向细胞的核周区域移动。由于ER-β复制位点可以从感染的细胞中分离出来，我建议在体外重建它们的MT相互作用。使用光学显微镜分析，我将首先研究与分离复制位点MT的结合，然后研究沿着MT的运动性。通过这种检测，我希望获得重要的新的洞察细胞以及可能的病毒因素的基础ER/MT的相互作用。最后，事实上，负端定向运动的复制位点不依赖于动力蛋白/动力蛋白复合物，表明一种新的微管电机机械需要确定。

Although microtubules (MTs) are known to play an important role in structural organization of the endoplasmic reticulum (ER) and ER-dynamics, relatively little is known about the molecular details underlying these interactions. The current proposal aims to use a novel in vitro system, based on mammalian cells, to study the molecular mechanisms of MT/ER interactions. It takes advantage of the observation that the cytoplasmic sites of vaccinia virus (vv) replication become entirely wrapped by the ER and these ER-enclosed sites move in a MT-dependent fashion towards the perinuclear region of the cell. Since the ER-enwrapped replication-sites can be isolated from infected cells, I propose to reconstitute their MT-interactions in vitro. Using a light microscopy assay, I will study first binding to, and then motility along MTs of isolated replication sites. With this assay I expect to gain important new insight into cellular as well as possibly viral factors underlying ER/MT interactions. Finally, the fact that the minus-end directed motility of the replication sites does not depend on the dynactin/dynein complex, suggests that a novel microtubule motor machinery needs to be identified.