Flagellar Glycoprotein Dynamics and Whole Cell Locomotion

单细胞藻类莱茵衣藻（Chlamydomonas reinhardtii）表现出两种不同的运动形式。研究得最好的形式是游泳，细胞一端的两个鞭毛像波浪一样跳动，导致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基质之间的接触点上滑动。该实验室先前的工作表明，这种滑动是一个精心调节的事件序列，需要鞭毛表面同时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。各种间接数据都有力地表明，高分子量鞭毛膜糖蛋白FMG-1在鞭毛的感觉（底物接触引发的跨膜信号传导）和运动（运动蛋白复合物活性与底物粘附位点的耦合）功能中起着核心作用。本研究的目的是利用分子和生物化学方法来了解FMG-1鞭毛膜糖蛋白的功能。为此，FMG-1基因将被克隆并测序。克隆FMG-1的基因编码将采用双管齐下的方法。首先，利用质谱获得的肽序列数据设计的寡核苷酸探针将用于两种PCR（聚合酶链反应）方法，从基因组DNA或鞭毛再生细胞的mRNA开始。在第二部分中，我们将使用一种亲和纯化的多克隆抗体来筛选衣藻表达文库，该多克隆抗体制备的合成肽的序列来自FMG-1糖蛋白，并且已被证明可以识别真正的蛋白质。

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.