Developmentally Regulated Calcium - Binding Proteins

发育调节钙结合蛋白

• 批准号: 9408635
• 负责人: Chandler Fulton
• 金额: $ 20万
• 依托单位: Brandeis University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9408635&HistoricalAwards=false
• 关键词: Developmentally; Regulated; Calcium; Binding; Proteins

9408635 Fulton Naegleria gruberi undergoes a developmental transformation from amoebae to flagellates over 100 minutes and may serve as a paradigm to study cell differentiation led us to calcium- binding proteins. Two calmodulins are synthesized during differentiation and become localized in flagellates, one in the flagella and the other in the flagellate cell body. Characterization of the single-copy intronless gene that encodes flagellar calmodulin (CaM-1) has shown that it cannot encode flagellate cell-body calmodulin (CaM-2). Initial attempts to isolate the CaM-2 gene led us to discover other differentiation-specific calcium-binding proteins, most notably centrin (a.k.a. caltractin). Our proposed research is focused on centrin and CaM-2. Centrin is a ubiquitous, diversely centriole-associated member of the calmodulin superfamily that has been implicated in Ca2+-mediated, ATPindependent contractility, a third contractile system. Although centrin is clearly a crucial eukaryotic protein, its functions, especially in relation to centrosome and basal body dynamics, are little understood. In Naegleria, centrin is developmentally regulated and becomes associated with the basal bodies when these organelles form de novo during differentiation. We plan to complete ongoing studies i) of the developmental regulation (e.g., phosphorylation) of centrin during Naegleria differentiation and basal-body assembly and ii) of remarkable structures formed by centrin-like proteins in certain other organisms, especially the spasmoneme of vorticellid ciliates. In order to better understand centrin, we plan iii) to seek and characterize proteins that associate with centrin using the cloned Naegleria centrin gene and the two-hybrid system in yeast and iv) to define by isolation, cloning and sequencing a centrin-related protein that is strongly implicated in contraction, the spasmin of Vorticella. In addition, we plan to define the second differentiation-specific calmodulin, CaM-2, by cloning its gene(s). These studies are of general interest in relation to two questions 1) What are the differences between the two differentiation-specific calmodulins of Naegleria, especially in relation to whether multiple calmodulins are a common feature among eukaryotes? 2) What is the structure and function of centrin, especially in Naegleria differentiation, what macromolecular structures does it form, and what is its relation to spasmin? Finally, a portion of our research effort continues to be directed toward obtaining DNA- mediated transformation of Naegleria, which would permit us to use the power of molecular genetics to dissect events during this otherwise ideal differentiation, such as the mechanism by which flagellar calmodulin (CaM-1) becomes localized in peninsular flagella. %%% This is a project which focuses on a group of protozoa on which very little work is currently being done, but which have evolved a fascinating interplay of cellular events. The protozoan, Naegleria gruberi, undergoes a dramatic shift from a crawling, amoeboid state to a swimming state by the de novo formation of flagella, the whip- like structures which motors many swimming cells. The work pursues this switch by concentrating on the new structural proteins which arise in the flagellated state, such as the contractile protein, centrin. In addition, by understanding the developmental control of genes which are newly transcribed, the switch to a new developmental pathway can be assessed. The switch may involve the expression of particular calcium binding proteins, or calmodulins. The project focuses on the characterization of the Naegleria centrin and the different forms of calmodulins. The association of centrins from Naegleria and another protozoan, Vorticella, with itself and other proteins is examined. The nature of this contractile protein is explored. The control of differential expression of calmodulins will be approached by testing the efficacy of obtai ning transgenic protists for these studies. The protists can then be transformed with different genetic variants of calmodulins and their effect on the development of flagella determined. By examining this unique organism, the diversity of microbes and their multiple ways of coping with their environment will be further illuminated. ***

9408635富尔顿 格鲁伯耐格里虫在100分钟内经历了从变形虫到鞭毛虫的发育转化，并可能作为研究细胞分化的范例，引导我们找到钙结合蛋白。两种钙调素在分化过程中合成，并定位于鞭毛中，一种在鞭毛中，另一种在鞭毛细胞体中。 对编码鞭毛钙调素（CaM-1）的单拷贝无内含子基因的表征表明，它不能编码鞭毛细胞体钙调素（CaM-2）。分离CaM-2基因的最初尝试使我们发现了其他分化特异性钙结合蛋白，最著名的是centrin（又名centrin）。caltractin）。我们的研究重点是centrin和CaM-2。中心蛋白是钙调素超家族中一个普遍存在的、与二聚体中心粒相关的成员，它参与了钙离子介导的、ATP非依赖性的收缩，这是第三种收缩系统。虽然中心蛋白是一个重要的真核生物蛋白质，但其功能，特别是与中心体和基体动力学的关系，知之甚少。在耐格里属中，中心蛋白受到发育调节，并且当这些细胞器在分化期间重新形成时，中心蛋白与基体相关联。我们计划完成正在进行的研究i）发育调节（例如，磷酸化）的中心蛋白在Naegleria分化和基体组装和ii）显着的结构形成的中心蛋白样蛋白在某些其他生物，特别是spasmoneme的vorticellid纤毛虫。 为了更好地理解中心蛋白，我们计划iii）使用克隆的耐格里酵母中心蛋白基因和酵母中的双杂交系统来寻找和表征与中心蛋白相关的蛋白，以及iv）通过分离、克隆和测序来定义与收缩强烈相关的中心蛋白相关蛋白，即涡孢菌的痉挛蛋白。此外，我们计划定义第二分化特异性钙调素，CaM-2， 通过克隆其基因。这些研究涉及两个问题：1）耐格里属的两种分化特异性钙调素之间的差异是什么，特别是关于多钙调素是否是真核生物的共同特征？2)中心蛋白的结构和功能是什么，特别是在耐格里属的分化中，它形成什么样的大分子结构，它与痉挛素的关系是什么？最后，我们的一部分研究工作继续致力于获得DNA介导的耐格里属转化，这将使我们能够利用分子遗传学的力量来剖析这种理想分化过程中的事件，例如鞭毛钙调蛋白（CaM-1）在半岛鞭毛中定位的机制。这是一个专注于一组原生动物的项目，目前对这组原生动物的研究很少，但它们已经进化出一种迷人的细胞事件相互作用。 原生动物格鲁伯耐格里虫通过鞭毛的重新形成，即驱动许多游泳细胞的鞭子状结构，经历了从爬行、变形虫状态到游泳状态的戏剧性转变。 这项工作通过集中研究在鞭毛状态下出现的新结构蛋白质，如收缩蛋白质，中心蛋白来实现这一转变。 此外，通过了解新转录的基因的发育控制，可以评估向新发育途径的转换。 这种开关可能涉及特定的钙结合蛋白或钙调素的表达。 该项目的重点是耐格里中心蛋白和不同形式的钙调素的表征。 协会的中心蛋白Naegleria和另一种原生动物，Vorticella，与本身和其他蛋白质的检查。这种收缩蛋白质的性质进行了探讨。 钙调素差异表达的控制将通过检测获得转基因原生生物的有效性来进行。 然后，可以用钙调素的不同遗传变体转化原生生物，并确定它们对鞭毛发育的影响。通过研究这种独特的有机体，微生物的多样性及其应对环境的多种方式将得到进一步阐明。 ***