Flagellar Glycoprotein Dynamics and Whole Cell Locomotion

鞭毛糖蛋白动力学和全细胞运动

• 批准号: 9808846
• 负责人: Robert Bloodgood
• 金额: $ 10万
• 依托单位: University of Virginia Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808846&HistoricalAwards=false
• 关键词: Flagellar; Glycoprotein; Dynamics; Whole; Cell

9808846 Bloodgood The unicellular alga, Chlamydomonas reinhardtii, exhibits two different forms of locomotion. The best-studied form is swimming, in which the wave-like beating of the two flagella at one end of the cell causes the cell to move through its aqueous environment. Far less well understood is the ability of Chlamydomonas to glide on a solid substratum. This gliding motility also involves the flagella, although the mechanism is quite different from swimming. The cell glides at the point of contact between the flagellar membrane outer surface and the solid substratum. Previous work from this laboratory suggests that this gliding is a carefully regulated sequence of events that requires the flagellar surface to exhibit both sensory and motor functions. This entails a signaling pathway, involving calcium ion regulation and protein phosphorylation and dephosphorylation, that couples the sensory and motor functions of the flagellar surface. A variety of circumstantial data strongly point to a high molecular weight flagellar membrane glycoprotein, FMG-1, as being central to both the sensory (substrate contact initiated transmembrane signaling) and motor (coupling of the activity of a motor protein complex to substrate adhesion sites) functions of the flagellum. The goal of the research project is to use coordinated molecular and biochemical approaches to understand the function of the FMG-1 flagellar membrane glycoprotein. To do this, the gene for FMG-1 will be cloned and sequenced.A two-pronged approach will be taken to cloning the gene coding for FMG-1. In the first, oligonucleotide probes, designed using peptide sequence data obtained by mass spectrometry, will be used in two PCR (Polymerase Chain Reaction) approaches, starting with genomic DNA or with mRNA from cells undergoing flagellar regeneration. In the second, a Chlamydomonas expression library will be screened using an affinity purified polyclonal antibody prepared against a synthetic peptide whose sequence comes from the FMG-1 glycoprotein and which has been shown to recognize the authentic protein.

9808846血好单细胞藻莱茵衣藻表现出两种不同的运动形式。研究最多的形式是游泳，细胞一端的两个鞭毛像波浪一样跳动，使细胞在水环境中移动。人们对衣藻在固体基质上滑行的能力知之甚少。这种滑行运动也涉及鞭毛，尽管其机制与游泳有很大不同。细胞在鞭毛膜外表面和固体底物之间的接触点滑动。该实验室以前的工作表明，这种滑行是一个精心调控的事件序列，需要鞭毛表面表现出感觉和运动功能。这需要一个信号通路，涉及钙离子调节和蛋白质磷酸化和去磷酸化，耦合鞭毛表面的感觉和运动功能。大量的环境数据有力地表明，一种高相对分子质量的鞭毛膜糖蛋白FMG-1对鞭毛的感觉(底物接触启动的跨膜信号传递)和运动(运动蛋白复合体的活动与底物粘附点的偶联)功能都是至关重要的。该研究项目的目标是使用协调的分子和生化方法来了解FMG-1鞭毛膜糖蛋白的功能。为此，FMG-1的基因将被克隆和测序。将采取双管齐下的方法来克隆FMG-1的编码基因。在第一种方法中，使用质谱仪获得的肽序列数据设计的寡核苷酸探针将用于两种PCR(聚合酶链式反应)方法，首先是基因组DNA，或者是经历鞭毛再生的细胞的mRNA。在第二个实验中，将使用针对合成肽的亲和纯化多克隆抗体来筛选衣原体表达文库，该合成肽的序列来自FMG-1糖蛋白，并已被证明识别真正的蛋白。