Flagellar Glycoprotein Dynamics and Whole Cell Locomotion

小行星9506230 整个细胞在固体基质上的运动涉及一系列精心设计的事件，需要细胞表面同时表现出感觉和运动功能，这些功能必须仔细整合。 披衣细胞沿着固体基质表现出滑动运动，这种形式的整个细胞运动依赖于鞭毛表面的活动。 这个实验室以前的工作表明存在一个信号通路（涉及c钙和蛋白磷酸化），耦合鞭毛表面的感觉和运动功能。 350 kD鞭毛膜糖蛋白的群体的交联导致钙内流，随后是60 kD鞭毛磷蛋白的去磷酸化，所述60 kD鞭毛磷蛋白通过其与350 kD糖蛋白的缔合结合到鞭毛膜的细胞质表面。 以一种尚未理解的方式，这些初始信号事件导致鞭毛运动蛋白复合物的激活，该复合物导致350 kD糖蛋白在鞭毛膜平面内的运动，从而引起运动。 在这个建议中概述的实验将扩展我们的理解鞭毛信号通路的感觉和运动事件在鞭毛表面耦合。 特别是，努力将集中在表征350 kD鞭毛膜糖蛋白和60 kD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使用插入诱变/标记获得的非滑动突变细胞系文库将筛选350 kD糖蛋白、60 kD磷蛋白和诱导60 kD磷蛋白去磷酸化的信号传导途径中的步骤中的缺陷。 这些突变体有望为克隆编码350 kD糖蛋白、60 kD磷蛋白和其他目前未知的信号通路信号组分的基因提供另一种方法。 将通过定向诱变克隆的DNA结合衣原体转化和转化体表征来进行350 kD鞭毛膜糖蛋白和60 kD磷蛋白的功能分析。 与理解该信号传导途径的分子方法，特别是迄今为止确定的两个关键参与者的功能平行，生物化学方法将被用来询问许多功能问题：1）60 kD磷蛋白是蛋白激酶吗？2)60 kD磷蛋白的磷酸化是否调节其功能和/或与350 kD膜糖蛋白的结合？3)350 kD糖蛋白上与60 kD磷蛋白结合的结合位点是什么？4）高等植物中的钙依赖蛋白激酶（CDPK）的衣原体鞭毛同源物是否在信号通路中起作用？ 将获得60 KD磷蛋白的单克隆抗体，并用于使用ELISA和Western印迹定量60 kD磷蛋白，通过免疫亲和层析纯化60 kD磷蛋白，用于表征非滑动细胞突变体和筛选λ gt 11表达文库。 这些实验结合了生物化学和分子生物学的方法，有望大大扩展我们对整个细胞运动调节的理解。 单细胞的绿色藻类，衣原体，是研究具有鞭状“尾巴”或鞭毛的细胞的滑行（不是游泳）运动的模型系统。本研究探讨了导致衣原体滑行运动的事件的协调性。 在鞭毛中发现的一种蛋白质复合体介导了滑翔运动。 该复合物是由一个较大的蛋白质含有共价连接的糖（350 kD糖蛋白）和一个较小的蛋白质含有共价连接的磷酸（60 kD磷蛋白）。 这些蛋白质的基因被克隆和测序。 在非滑行衣原体的现有突变体文库中鉴定了基因的突变形式。 这些突变体库也是鞭毛滑动信号机制中重要基因的来源。 这些基因的突变形式也将在衣原体中合成和重新表达。 对介导滑动运动的蛋白质进行生物化学分析，以确定蛋白质的生物化学功能。 60 kD磷蛋白将被纯化，并回答以下问题：它是否磷酸化鞭毛的组分？ 它所显示的磷酸化是其活性所必需的吗？它如何与350 kD糖蛋白相互作用？ 其他蛋白质已知是信号介质，也发现在衣原体将被确定为候选人协调的一部分滑行运动。 这项工作是关于细胞运动机制以及细胞马达如何协调产生运动的更大问题的一部分。 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。 ***

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. ***