Flagellar Glycoprotein Dynamics and Whole Cell Locomotion

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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.