Flagellar Glycopro#ein Dynamics and Whole Cell Locomotion

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Active movement of a class of 350 kDa glycoproteins within the plane of the Chlamydomonas flagellar membrane is responsible for whole cell gliding motility in this organism. Plasma membrane protein dynamics may also be involved in many other forms of whole cell locomotion along solid substrates or extracellular matrix. Contact of the flagellar membrane with a solid substrate is hypothesized to initiate a transmembrane signaling pathway that activates an intraflagellar motor responsible for binding to and applying force to the flagellar membrane glycoproteins. Crosslinking of certain populations of flagellar membrane glycoproteins with anti-carbohydrate monoclonal antibodies is an artificial means to initiate the signaling pathway (involving calcium influx and changes in protein phosphorylation) and result in redistribution of the flagellar membrane proteins recognized by the antibodies. The present study is directed towards an elucidation of the role of protein phosphorylation in the signaling pathway and the identification and characterization of the biological motor responsible for glycoproteins movements and hence whole cell gliding motility. Studies will address the regulated association of flagellar membrane proteins and protein kinases with the 350 Kda flagellar membrane glycoproteins, and the association of phosphoproteins and other proteins in the membrane-matrix fraction with axonemal and brain microtubules. An in vitro system for studying flagellar membrane dynamics using inside-out flagellar membrane vesicles and purified microtubules will be developed. Biological motor activity will be sought within the membrane-matrix compartment using in vitro microtubule gliding assays. The 3S calcium-specific ATPase in the membrane -matrix fraction will be purified and characterized as a candidate motor. Monoclonal antibodies to various flagellar membrane-matrix phosphoproteins and to candidate motor proteins will be obtained for use as probes for studying the role of these proteins in flagellar signaling and force production for glycoprotein movement and whole cell locomotion. %%% The unicellular green alga, Chlamydomonas, is a widely used model organism for studying a wide variety of biological questions. The alga has flagellae, which enable it to swim freely in aqueous media, in a manner which is functionally similar to other flagellar-propelled motilities in other organisms. The Chlamydomonas flagellum has therefore been a highly productive model for studying the mechanism of flagellar motility in general. In addition to the free-swimming mode of locomotion, Chlamydomonas also exhibits a gliding motility on solid substrata, which also involves the flagellum, or, more specifically, the plasma membrane surrounding the flagellum. The mechanism of this gliding motility has been traced to the active movement of certain membrane glycoproteins within the plane of the flagellar membrane. It is likely that this system will serve as a good general model for studying "deliberate" movements of membrane proteins within the plane of the membrane.