Cell Cycle Regulation of Microtubule Assembly

9315700以上建议的研究是关于微管的组装和功能在细胞周期中是如何被调节的。将使用多学科的方法来检验这一假设，即77kD棘皮MT相关蛋白(EMAP)是多亚基G蛋白复合体的一部分，其功能受磷酸化和GTP结合的调节。生物化学方法将用于鉴定EMAP结合蛋白，直接光亲和标记将确定这些蛋白中是否有任何是GTP结合蛋白。免疫学技术将用于表征EMAP-Dynamin相互作用的体外免疫沉淀法和体内免疫金标记的电子显微镜。在体内，EMAP和Dynamin的磷酸化将通过32-P标记和免疫沉淀完成。微管组装动力学将通过视频增强差示对比光镜检查免疫耗竭的间期和有丝分裂提取物。亲和纯化的抗动力素和抗EMAP抗体将被显微注射到海胆卵裂球中，以确定它们对细胞分裂的影响。杆状病毒中的基因工程EMAP构建体将被导入昆虫细胞，并检测它们对内源微管和细胞器阵列的影响。这项拟议研究的完成将为有丝分裂过程中细胞形态发生的调控提供洞察力。%微管是真核细胞骨架必不可少的动态组件，是鞭毛和纤毛跳动(精子或原生动物游泳、支气管粘液移动等现象所必需)、粒子和囊泡从细胞一端到另一端的运动(例如，含有神经递质的突触体从神经细胞的细胞体向下包装神经递质的突触体，一直到轴突顶端，内容通过胞吐释放)和细胞分裂期间子代染色体分离等极其重要的细胞和细胞内运动现象的媒介。微管在空间组织、功能能力、长度和稳定性方面的调节还不是很清楚，但显然对细胞的正常功能至关重要。已知某些蛋白质与微管相关，并被称为MAP(微管相关蛋白)；它们被认为参与了微管相关功能的调节或调节。以无脊椎动物细胞为模型，在海胆中发现了一个新的MAP(称为EMAP)，经过分析，它与已知的G-蛋白的调节亚基相似(G-蛋白是细胞中的一类调节蛋白，参与了广泛的信号转导现象)，并被证明与Dynamin结合，Dynamin是一种GTP-ase，与某些微管介导的囊泡运动现象有关。基于这些发现，已经开发了一个可测试的模型，其中Dynamin和EMAP被假设为相互关联的微管连接蛋白，与微管和囊泡相关联。这些关联又可能受GTP结合和磷酸化的调节。本项目中提出的实验将测试模型的关键部分。这项研究有望阐明微管系统在细胞中的工作原理。***

9315700 Suprenant The proposed research is an investigation of how microtubule assembly and function is regulated during the cell cycle. A multidisciplinary approach will be used to test the hypothesis that a 77 kD echinoderm MT-associated protein (EMAP) is part of a multi-subunit G-protein complex whose function is regulated by phosphorylation and GTP- binding. Biochemical methods will be used to identify EMAP- binding proteins and direct photoaffinity labeling will determine whether any of these proteins are GTP-binding proteins. Immunological techniques will be used to characterize EMAP-dynamin interactions in vitro by immunoprecipitation and in vivo by immunogold labeling for electron microscopy. In vivo phosphorylation of EMAP and dynamin will be accomplished by 32-P labeling followed by immunoprecipitation. Microtubule assembly dynamics will be examined in immunodepleted interphase and mitotic extracts by video-enhanced differential contrast light microscopy. Affinity-purified anti-dynamin and anti-EMAP antibodies will be microinjected into sea urchin blastomeres to determine their effects on cell division. Genetically engineered EMAP constructs in baculovirus will be transfected into insect cells and examined for their effects on endogenous microtubule and organelle arrays. The completion of the proposed study will provide insight into the regulation of cellular morphogenesis during mitosis. %%% Microtubules are essential and dynamic components of the eukaryotic cytoskeleton, and are the mediators of such critically important cellular and intracellular motility phenomena as flagellar and ciliary beating (necessary for such phenomena as spermatozoan or protozoan swimming, movement of bronchial mucus, etc.), movement of particles and vesicles from one end of the cell to another (e.g., movement of synaptosomes containing neurotransmitters from the cell body of a nerve cell, where the synaptosomes are packaged, down to the tip of the axon, where t he contents are released by exocytosis) and separation of daughter chromosomes during cell division. The regulation of microtubules, in terms of their spatial organization, functional capabilities, and length and stability is not yet well understood, yet obviously is critical to the proper functioning of the cell. Certain proteins are known to be associated with microtubules, and are termed MAPs (microtubule-associated proteins); these are presumed to be involved in the regulation or mediation of microtubule- associated functions. Working with invertebrate cells as models, a novel MAP (termed EMAP) was found in sea urchins which, upon analysis, was shown to be similar to known regulatory subunits of G-proteins (a class of regulatory proteins in cells which are involved in a wide variety of signal transduction phenomena) and which was also shown to bind to dynamin, a GTP-ase implicated in certain microtubule- mediated vesicle movement phenomena. Based on these findings, a testable model has been developed in which dynamin and EMAP are postulated to be microtubule linker proteins that associate with each other, with microtubules, and with vesicles. These associations may in turn be regulated by GTP binding and phosphorylation. The experiments proposed in this project will test key parts of the model. The research promises to shed light on how the microtubule system works in cells. ***