The objective is to define the molecular events in the signal transduction by which the ciliated protozoan paramecium tetraurelia responds to stimuli by changing its swimming speed and direction. These events include regulation of ion channels and regulation of the ciliary axoneme's motility. Paramecium combines the experimental advantages of a microbe (genetics and biochemistry) with the advantages of a large size (electrophysiology and microinjection). Ca and cyclic nucleotides modulate changes in ciliary beta frequency and stroke orientation. Many of the proteins (Ca channels, calmodulin, calmodulin-binding proteins, adenylate cyclase, protein kinases, phosphatases, dyneins) essential to this process are localized in the cilium. Some of these proteins have already been purified from Paramecium; others are currently being purified. Antibodies (polyclonal and monoclonal) against purified proteins will be used to test definitively whether a given protein functions in the sensory pathway. Methods will be developed for adding antibodies to living cells by microinjection, electroporation or reversible permeabilization. The effects of each antibody on the swimming speed and direction will be assessed. Monoclonal antibodies raised against semi-purified fractions (e.g., minor proteins of ciliary membrane which should include the Ca channel proteins) will be screened for their effects on the swimming behavior of paramecia. Any antibodies that show behavioral effects will be assayed electrophysiologically (in collaboration with Ching Kung's laboratory) to determine which, if any, of the ion conductance mechanisms known to function in sensory transduction is affected by each. Antibodies found to alter electrical properties of the membrane in voltage-clamp studies in whole cells will be characterized further by studying single channels in isolated patch-clamped surface membrane. These studies may lead to the identification of proteins that form ion channels or regulate their activities. To study the targets of protein phosphorylation, specific proteins will be immunoprecipitated from detergent extracts of quick-killed cells, and their states of phosphorylation will be compared in cells stimulated to swim fast or backwards, or in wild type and mutant cells. Antibodies will also be used to screen behavioral mutants for enzymic differences, and to screen expression libraries of Paramecium DNA in E. coli.
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