MCB9506838 Paul Sammak The regulatory mechanisms that convert a stationary, quiescent cell into an active motile cell are not known. Confluent cells in culture, made quiescent by serum starvation and contact inhibition, can be induced to move by mechanically denuding a zone for cells to move into. Scraping does more than provide space for cell movement since we have found that specific signals are derived from injured cells to initiate motility. The injury produced a brief wave of elevated intracellular free Ca2+ ( Ca2+ i) that propagated for a limited distance through the monolayer. The transient elevation of Ca2+ i determined the rate of motility for hours after motility was initiated. We also have pharmacological evidence that intracellular free cyclic AMP ( cAMP i) is modulated during scrape injury and that elevation of cAMP i can either stimulate or inhibit movement depending on the time of treatment. It is not known how the transient Ca2+ i and cAMP i -- dependent signals might control the long term rate of cell locomotion. The requirement for changes of cAMP i and Ca2+ i during relief from contact inhibition in confluent monolayers will be measured. The requirement for these intracellular messengers for the maintenance of steady state cell motility will also be examined. I. Measure the Ca2+ i elevation induced by release from contact inhibition by fura-2 and BTC fluorescence ratio imaging and determine whether the Ca2+ i wave is necessary and sufficient for stimulation of motility. Necessity will be tested by blocking Ca2+ influx and stores release. Sufficiency will be tested by artificially inducing Ca2+ i elevations with Nitr-5. II. Measure cAMP i dynamics induced by release from contact inhibition and determine whether changes in cAMP i are necessary and sufficient for stimulation of motility. Cyclic AMP will be measured by fluorescence ratio imaging of the cAMP i indicator, FlCRhR (Fluorescein Catalytic, Rhodamine Regulatory, pronounced flicker), the fluorescently derivatized cAMP dependent protein kinase. If endogenous changes are discovered, they will be perturbed with the inhibitor, RpcAMPS and the stimulator, SpcAMPS. We have evidence that one of the few known functions for the Ca2+ i wave is the stimulation of wound healing. We have shown that Ca2+ influx is necessary for the full rate of cell motility, but it is not known whether Ca2+ i mobilization is sufficient to induce motility or whether release of Ca2+ i stores is necessary for motility. The role of cAMP i in motility is less certain than that of Ca2+ i. Our knowledge has been hampered by cAMP detection methods that require biochemical averages of millions of cells. Only recently has a method been developed to measure dynamics of cAMP i in single living cells. There have been no direct determinations of whether cells normally modulate cAMP i to control whole cell locomotion, although we have indirect evidence that they might. If our hypothesis is correct, we will acquire significant insight into the second messenger control of cell motility. %%% This project will investigate the intracellular messages that change cell behavior and initiate cell motility. It is known that Ca2+ i and cAMP i -are messengers that activate enzymes within the cell and can change cell behavior. This project will measure both Ca2+ i and cAMP i in single living cells as they begin to move. Changes in the concentration of Ca2+ and cAMP will be measured to see if cells make use of these messengers as they begin to move. These changes will then be blocked to see if they were necessary for cell movement and will artificially induce these changes to see if these messengers are sufficient in themselves to make cells move, or whether other signals might be required at the same time. The role of cAMP i in motility is less certain than that of Ca2+ i. Acquisition of knowledge has been hampered by cAMP detection methods that require biochemical averages of millions of cells. Only recently has a method been developed to measure dynamics of cAMP i in single living cells. There have been no direct determinations of whether cells normally modulate cAMP i to control whole cell locomotion, although indirect evidence that they might. This project will provide insight into the control of cell motility by second messengers. ***
