9506230 Bloodgood Whole cell locomotion alone a solid substrate involves a carefully choreographed sequence of events that requires the cell surface to exhibit both sensory and motor functions, which must be carefully integrated. Chlamydomonas exhibits gliding motility along a solid substrate, and this form of whole cell locomotion is dependent upon the activities of the flagellar surface. Previous work from this laboratory suggests the existence of a signaling pathway (involving c calcium and protein phosphorylation) that couples the sensory and motor functions of the flagellar surface. Crosslinking of a population of 350 kD flagellar membrane glycoproteins results in calcium influx followed by the dephosphorylation of a 60 kD flagellar phosphoprotein that binds to the cytoplasmic surface of the flagellar membrane through its association with the 350 kD glycoprotein. In a manner not yet understood, these initial signaling events result in activation of a flagellar motor protein complex that derives the movement of the 350 kD glycoproteins within the plane of the flagellar membrane thereby bringing about locomotion. The experiments outlined in this proposal will extend our understanding of the flagellar signaling pathway by which sensory and motor events at the flagellar surface are coupled. In particular, efforts will be focused on characterizing the functions performed by the 350 kD flagellar membrane glycoprotein and the 60 kD flagellar phosphoprotein. These proteins will be cloned sequenced. A library of non-gliding mutant cell lines obtained by Kozminski and Rosenbaum using insertional mutagenesis/tagging will be screened for defects in the 350 kD glycoproteins, the 60 kD phosphoprotein, and steps in the signaling pathway that induces the dephosphorylation of the 60 kD phosphoprotein. These mutants hold the promise of providing an alternative approach to cloning genes encoding the 350 kD glycoprotein, the 60 kD phosphoprotein, and other, curren tly unknown, components in the signal components of the signaling pathway. Functional analysis of the 350 kD flagellar membrane glycoprotein and the 60 kD phosphoprotein will be performed by directed mutagenesis of cloned DNAs coupled with transformation of Chlamydomonas and characterization of the transformants. In parallel with the molecular approaches to understanding this signaling pathway and, in particular, the function of the two key players identified to date, biochemical approaches will be utilized to ask a number of functional questions: 1) Is the 60 kD phosphoprotein a protein kinase? 2) Does the phosphorylation of the 60 kD phosphoprotein regulate its function and/or its association with he 350 kD membrane glycoprotein? 3) What is the binding site on the 350 kD glycoprotein to which 60 kD phosphoprotein binds? and 4) Does the Chlamydomonas flagellar homologue of the calcium dependent protein kinase (CDPK) from higher plants play a role in the signaling pathway? Monoclonal antibodies to the 60 KD phosphoprotein will be obtained and utilized for quantitating the 60 kD phosphoprotein using ELISA and Western blots, purifying the 60 kD phosphoprotein by immunoaffinity chromatography, for characterizing the non-gliding cell mutants and for screening a lambda gt11 expression library. These experiments, utilizing a combining of biochemical and molecular approaches, hold the promise of greatly extending our understanding of the regulation of whole cell locomotion. %%% The unicellular green alga, Chlamydomonas, is a model system for studying gliding (not swimming) motion of cells with a whiplike "tail" or flagellum. This study explores the coordination of events which bring about the gliding motion in Chlamydomonas. A complex of proteins found within the flagellum mediates the gliding motion. The complex is comprised of a larger protein containing covalently-attached sugars (350 kD glycoprotein) and a smaller protein containing covalently attached pho sphates (60 kD phosphoprotein). The genes for these proteins are cloned and sequenced. Mutant forms of the genes are identified in existing mutant libraries of non-gliding Chlamydomonas. These mutant libraries are also the source of genes important in the signaling mechanisms involved in flagellar gliding. Mutant forms of the genes will also be synthesized and re-expressed in Chlamydomonas. Biochemical analysis of proteins which mediate the gliding motion are pursued to identify the biochemical function of the proteins. the 60 kD phosphoprotein will be purified and the following questions answered: Does it phosphorylate components of the flagella? Is the phosphorylation which it shows necessary for its activity? How does it interact with the 350 kD glycoprotein? Other proteins known to be signaling mediators and also found in Chlamydomonas will be identified as candidates for coordinating a part of the gliding motion. This work is part of a larger question about the mechanism of cell motility and how cellular motors are coordinated to produce motion. Application for this work could be found in the area of how molecular machines are coordinated and synthesized. ***
