Flagellar Glycoprotein Dynamics and Whole Cell Locomotion

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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.