Cell Cycle Regulation of Microtubule Assembly

微管组装的细胞周期调控

• 批准号: 9315700
• 负责人: Kathy Suprenant
• 金额: $ 57.3万
• 依托单位: University of Kansas Main Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9315700&HistoricalAwards=false
• 关键词: Cell; Cycle; Regulation; Microtubule; Assembly

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. ***

9315700 Suprenant提出的研究是对细胞周期中如何调节微管组装和功能的调查。多学科方法将用于验证77 kD棘皮mt相关蛋白（EMAP）是多亚基g蛋白复合物的一部分，其功能受磷酸化和GTP结合调节的假设。生化方法将用于鉴定EMAP结合蛋白，直接光亲和标记将确定这些蛋白是否为gtp结合蛋白。免疫学技术将被用于表征emap -动力蛋白相互作用，在体外通过免疫沉淀和体内通过免疫金标记的电子显微镜。在体内，EMAP和动力蛋白的磷酸化将通过32-P标记和免疫沉淀来完成。微管组装动力学将在免疫缺失的间期和有丝分裂提取物中通过视频增强差分对比光学显微镜进行检查。将亲和纯化的抗动力蛋白和抗emap抗体微注射到海胆卵裂球中，以确定它们对细胞分裂的影响。杆状病毒的基因工程EMAP构建体将被转染到昆虫细胞中，并检测其对内源性微管和细胞器阵列的影响。该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见解。微管是真核细胞骨架的基本和动态组成部分，是一些至关重要的细胞和细胞内运动现象的介质，如鞭毛和纤毛跳动（精子或原生动物游泳、支气管粘液运动等现象所必需的）、颗粒和囊泡从细胞一端到另一端的运动（例如，神经细胞胞体中含有神经递质的突触体的运动）、突触体被包装的地方，一直到轴突的尖端，在那里，内容物通过胞外分泌释放出来，在细胞分裂过程中，子染色体分离。微管在空间组织、功能能力、长度和稳定性方面的调控尚不清楚，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，称为MAPs（微管相关蛋白）；这些被认为参与了微管相关功能的调节或调解。以无脊椎动物细胞为模型，在海胆中发现了一种新的MAP（称为EMAP），经分析，它与已知的g蛋白（细胞中的一类调节蛋白，参与各种信号转导现象）的调节亚基相似，并且还被证明与动力蛋白结合，动力蛋白是一种gtp酶，涉及某些微管介导的囊泡运动现象。基于这些发现，已经开发了一个可测试的模型，其中假定动力蛋白和EMAP是微管连接蛋白，它们与微管和囊泡相互关联。这些关联可能反过来受到GTP结合和磷酸化的调节。本项目提出的实验将测试模型的关键部分。这项研究有望揭示微管系统在细胞中的工作原理。＊＊＊