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方式。其中最著名的是游泳，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，但其机制与游泳非常不同。细胞在鞭毛膜外表面和固体基质之间的接触点上滑动。布拉德古德博士实验室之前的工作表明，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。该项目的长期目标是利用分子和生化方法来了解FMG-1鞭毛膜糖蛋白的功能。利用肽抗体筛选衣藻cDNA表达文库，获得了克隆插入片段，有望用于从衣藻基因组文库中克隆全长基因。退化寡核苷酸（从FMG-1肽序列设计）也将用于基于RT-PCR的克隆和筛选衣藻细菌人工染色体（BAC）文库。将筛选现有的在滑动运动中有缺陷的插入突变体，以确定它们中是否有FMG-1基因缺陷；如果是这样，这些突变体将用于体内功能研究，如有必要，用于克隆标记的FMG-1基因。一旦FMG-1基因被克隆和测序，将进行转化研究，以过度表达该基因的正常或突变版本，以确定关键功能域和评估功能。预计其中一些基因结构将作为显性负突变发挥作用，干扰内源性FMG-1的正常功能。另一种方法是表征FMG-1与其他蛋白质的相互作用。鞭毛磷蛋白（pp60）先前被确定为FMG-1的结合伙伴，各种体内和体外方法将用于表征pp60和FMG-1之间的相互作用。将确定pp60的FMG-1结合域，并测试磷酸化在相互作用中的作用。这些在体内和体外的方法有望为主要鞭毛膜糖蛋白的功能和模型单细胞生物滑翔运动的机制提供有用的信息，具有很大的实验优势。人们对衣藻细胞在基质上滑动的原因知之甚少；这些研究可能揭示了有关原生鞭毛一般滑翔行为的有趣信息。这些研究也可能为膜蛋白的功能和功能之间的关系提供意想不到的新见解。

The unicellular alga, Chlamydomonas reinhardtii, exhibits two different kinds of locomotion. The best known of these 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, but the mechanism is very different from swimming. The cell glides at the point of contact between the flagellar membrane outer surface and the solid substratum. Previous work from Dr. Bloodgood's 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 long-term goal of the project is to use molecular and biochemical approaches to understand the function of the FMG-1 flagellar membrane glycoprotein. Screening of a Chlamydomonas cDNA expression library using a peptide antibody has yielded cloned inserts that promise to be useful for cloning the full-length gene from a Chlamydomonas genomic library. Degenerate oligonucleotides (designed from FMG-1 peptide sequences) will also be used, both for RT-PCR based cloning and to screen a Chlamydomonas Bacterial Artificial Chromosome (BAC) library. An existing collection of insertional mutants that are defective in gliding motility will be screened to determine if any of them possess a defect in the gene for FMG-1; if so, these mutants will be used for in vivo functional studies and, if necessary, for cloning the tagged FMG-1 gene. Once the FMG-1 gene is cloned and sequenced, transformation studies will be carried out to overexpress either normal or mutant versions of the gene, in order to identify key functional domains and assess function. It is anticipated that some of these gene constructs will function as dominant negative mutations to interfere with the normal function of endogenous FMG-1. Another approach is to characterize the interactions of FMG-1 with other proteins. A flagellar phosphoprotein (pp60) was previously identified as a binding partner for FMG-1, and a variety of in vivo and in vitro approaches will be used to characterize the interaction between pp60 and FMG-1. The FMG-1 binding domain for pp60 will be determined and the role of phosphorylation in the interaction will be tested.These in vivo and in vitro approaches are expected to provide useful information about the function of the major flagellar membrane glycoprotein and the mechanism of gliding motility in a model unicellular organism that offers great experimental advantages. Little is known about why Chlamydomonas cells glide over substrata; these studies may reveal interesting information about protistan flagellar gliding behavior in general. These studies may also provide unexpected new insights into functions of and functional relationships between membrane proteins in general.