Flagellar Glycoprotein Dynamics and Whole Cell Locomotion

鞭毛糖蛋白动力学和全细胞运动

• 批准号: 9506230
• 负责人: Robert Bloodgood
• 金额: $ 34.5万
• 依托单位: University of Virginia Main Campus
• 依托单位国家: 美国
• 项目类别: Continuing 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=9506230&HistoricalAwards=false
• 关键词: Flagellar; Glycoprotein; Dynamics; Whole; Cell

9506230 Bloodgood Whole cell locomotion alone a solid substrate involves a carefully choreographed sequence of events that requires the cell surface to exhibit both sensory and motor functions, which must be carefully integrated. Chlamydomonas exhibits gliding motility along a solid substrate, and this form of whole cell locomotion is dependent upon the activities of the flagellar surface. Previous work from this laboratory suggests the existence of a signaling pathway (involving c calcium and protein phosphorylation) that couples the sensory and motor functions of the flagellar surface. Crosslinking of a population of 350 kD flagellar membrane glycoproteins results in calcium influx followed by the dephosphorylation of a 60 kD flagellar phosphoprotein that binds to the cytoplasmic surface of the flagellar membrane through its association with the 350 kD glycoprotein. In a manner not yet understood, these initial signaling events result in activation of a flagellar motor protein complex that derives the movement of the 350 kD glycoproteins within the plane of the flagellar membrane thereby bringing about locomotion. The experiments outlined in this proposal will extend our understanding of the flagellar signaling pathway by which sensory and motor events at the flagellar surface are coupled. In particular, efforts will be focused on characterizing the functions performed by the 350 kD flagellar membrane glycoprotein and the 60 kD flagellar phosphoprotein. These proteins will be cloned sequenced. A library of non-gliding mutant cell lines obtained by Kozminski and Rosenbaum using insertional mutagenesis/tagging will be screened for defects in the 350 kD glycoproteins, the 60 kD phosphoprotein, and steps in the signaling pathway that induces the dephosphorylation of the 60 kD phosphoprotein. These mutants hold the promise of providing an alternative approach to cloning genes encoding the 350 kD glycoprotein, the 60 kD phosphoprotein, and other, curren tly unknown, components in the signal components of the signaling pathway. Functional analysis of the 350 kD flagellar membrane glycoprotein and the 60 kD phosphoprotein will be performed by directed mutagenesis of cloned DNAs coupled with transformation of Chlamydomonas and characterization of the transformants. In parallel with the molecular approaches to understanding this signaling pathway and, in particular, the function of the two key players identified to date, biochemical approaches will be utilized to ask a number of functional questions: 1) Is the 60 kD phosphoprotein a protein kinase? 2) Does the phosphorylation of the 60 kD phosphoprotein regulate its function and/or its association with he 350 kD membrane glycoprotein? 3) What is the binding site on the 350 kD glycoprotein to which 60 kD phosphoprotein binds? and 4) Does the Chlamydomonas flagellar homologue of the calcium dependent protein kinase (CDPK) from higher plants play a role in the signaling pathway? Monoclonal antibodies to the 60 KD phosphoprotein will be obtained and utilized for quantitating the 60 kD phosphoprotein using ELISA and Western blots, purifying the 60 kD phosphoprotein by immunoaffinity chromatography, for characterizing the non-gliding cell mutants and for screening a lambda gt11 expression library. These experiments, utilizing a combining of biochemical and molecular approaches, hold the promise of greatly extending our understanding of the regulation of whole cell locomotion. %%% The unicellular green alga, Chlamydomonas, is a model system for studying gliding (not swimming) motion of cells with a whiplike "tail" or flagellum. This study explores the coordination of events which bring about the gliding motion in Chlamydomonas. A complex of proteins found within the flagellum mediates the gliding motion. The complex is comprised of a larger protein containing covalently-attached sugars (350 kD glycoprotein) and a smaller protein containing covalently attached pho sphates (60 kD phosphoprotein). The genes for these proteins are cloned and sequenced. Mutant forms of the genes are identified in existing mutant libraries of non-gliding Chlamydomonas. These mutant libraries are also the source of genes important in the signaling mechanisms involved in flagellar gliding. Mutant forms of the genes will also be synthesized and re-expressed in Chlamydomonas. Biochemical analysis of proteins which mediate the gliding motion are pursued to identify the biochemical function of the proteins. the 60 kD phosphoprotein will be purified and the following questions answered: Does it phosphorylate components of the flagella? Is the phosphorylation which it shows necessary for its activity? How does it interact with the 350 kD glycoprotein? Other proteins known to be signaling mediators and also found in Chlamydomonas will be identified as candidates for coordinating a part of the gliding motion. This work is part of a larger question about the mechanism of cell motility and how cellular motors are coordinated to produce motion. Application for this work could be found in the area of how molecular machines are coordinated and synthesized. ***

9506230血缘良好的全细胞运动仅靠固体底物就需要精心设计的一系列事件，这要求细胞表面既表现出感觉功能又表现出运动功能，这两种功能必须仔细整合。衣藻表现出沿固体底物的滑动运动，这种形式的全细胞运动依赖于鞭毛表面的活动。该实验室以前的工作表明，鞭毛表面存在一条信号通路(涉及c、钙和蛋白质磷酸化)，使鞭毛表面的感觉和运动功能耦合。350kD鞭毛膜糖蛋白的交联导致钙内流，随后60kD鞭毛磷蛋白通过与350kD糖蛋白的结合而结合到鞭毛膜的细胞质表面。以一种尚不清楚的方式，这些最初的信号事件导致鞭毛运动蛋白复合体的激活，该复合体导致350kD糖蛋白在鞭毛膜平面内的运动，从而带来运动。这项提议中概述的实验将扩大我们对鞭毛信号通路的理解，通过该通路，鞭毛表面的感觉和运动事件是耦合的。特别是，将重点研究350kD鞭毛膜糖蛋白和60kD鞭毛磷蛋白所执行的功能。这些蛋白质将被克隆测序。Kozminski和Rosenbaum通过插入突变/标记获得的非滑动突变细胞系的文库将被筛选，以寻找350kD糖蛋白、60kD磷酸蛋白的缺陷以及诱导60kD磷酸蛋白去磷酸化的信号通路中的步骤。这些突变体有望提供一种替代方法来克隆编码350kD糖蛋白、60kD磷酸蛋白以及信号通路信号成分中其他目前未知的成分的基因。350kD鞭毛膜糖蛋白和60kD磷酸蛋白的功能分析将通过克隆DNA的定向突变和衣藻转化并对转化子进行鉴定。与理解这一信号通路的分子方法，特别是迄今已发现的两个关键角色的功能的平行，生化方法将被用来提出一些功能问题：1)60kD的磷酸蛋白是一种蛋白激酶吗？2)60kD的磷酸化是否调节其功能和/或其与350kD的膜糖蛋白的关系？3)60kD的磷蛋白与350kD的糖蛋白结合的结合部位是什么？以及4)来自高等植物的钙依赖蛋白激酶(CDPK)的鞭毛同源物是否在信号通路中发挥作用？我们将获得60kD磷酸化蛋白的单抗，并利用ELISA和Western blotts对60kD磷酸化蛋白进行定量，免疫亲和层析纯化60kD磷化蛋白，鉴定非滑动细胞突变体，筛选lambda gt11表达文库。这些实验，利用生物化学和分子方法的结合，有望极大地扩展我们对整个细胞运动调节的理解。单细胞绿藻衣藻是研究有鞭毛的细胞滑行(而不是游动)运动的模型系统。本研究探讨导致衣藻滑行运动的事件间的协调性。在鞭毛中发现的一种蛋白质复合体参与了滑行运动。该复合体由一个较大的含有共价连接的糖的蛋白质(350kD糖蛋白)和一个较小的含有共价连接的磷酸的蛋白质(60kD的磷酸蛋白)组成。这些蛋白质的基因被克隆和测序。这些基因的突变形式在现有的非滑动衣藻突变文库中得到了鉴定。这些突变文库也是鞭毛滑动信号机制中重要基因的来源。基因的突变形式也将被合成并在衣藻中重新表达。对调节滑行运动的蛋白质进行生化分析，以确定蛋白质的生化功能。60kD的磷酸蛋白将被提纯，并回答以下问题：它是否使鞭毛的组成部分磷酸化？它所显示的磷酸化是其活性所必需的吗？它如何与350kD的糖蛋白相互作用？在衣藻中也发现了其他已知的信号介体蛋白，它们将被确定为协调部分滑行运动的候选蛋白。这项工作是一个更大的问题的一部分，这个问题涉及细胞运动的机制以及细胞马达是如何协调产生运动的。这项工作的应用可以在分子机器如何协调和合成的领域找到。***