Our objective is to understand in molecular terms the process by which a eucaryotic cell perceives an external stimulus and responds by changing its motile behavior. The ciliated protozoan Paramecium tetraurelia adjusts its swimming speed and direction in response to various stimuli. Paramecium combines the advantages of a microbe for genetic and biochemical studies with the advantages of large size for electrophysiological and cell biological studies including microinjection. Our combined electrophysiological, genetic, and biochemical studies give circumstantial evidence for the involvement of specific proteins in the stimulus-response path. We will raise antibodies (polyclonal and monoclonal) against these proteins, and use them as specfic probes to test definitively whether a given protein (antigen) functions in the sensory pathway. Three types of antigens are available: 1) purified Paramecium proteins (a Ca-ATPase, immobilization antigen, calmodulin, ciliary protein kinase), 2) semi-purified protein mixtures known to contain components that function in the regulation of behavior (ciliary membrane proteins, cytoplasmic proteins which """"""""cure"""""""" behavioral mutants when microinjected), and 3) peptides identified on SDS-polyacrylamide gels as different in wild type cells and behavioral mutants, or as covalently altered in stimulated cells. The purified proteins will be used directly to raise rabbit or mouse antiserum. Alternatively, monoclonal antibodies raised against impure antigens will be screened for their affects on the swimming behavior of paramecia when applied externally or microinjected. Antibodies which show behavioral effects will be assayed electrophysilogically to determine which, if any, of the ion conductance mechanisms known to function in sensory transduction is affected by each antibody or antiserum. The effects of antibodies upon in vitro ion fluxes, measured in ciliary membrane vesicles and in planar lipid bilayers, will be assessed. Immunohistochemistry at the light and electron microscopic levels will show where a given antigen is located in the cell. Affinity chromatography with immobilized antibody will be used to purify components of membrane, axoneme, or cytoplasm that function in the stimulus-response pathway. In short we hope to use antibodies as specific, high affinity probes of the stimulus-response path in Paramecium, as neurotoxins have been used in studies of vertebrate sensory paths.
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