The basis of how cells detect water stress remains largely obscure and opportunities to study its molecular mechanism are few. Upon hyper-osmotic shock, yeast cells release Ca2+ from their vacuoles to the cytoplasm. We cloned the gene that encodes a cation channel in the vacuolar membrane, Yvclp, that is apparently the conduit for this Ca2+ release. We recently found that physical pressures or osmotic upshocks open this channel under patch clamp, making it possible that the conduit is also the sensor of the force in vivo. This hypothesis will be tested. We will change Yvclp by mutations or use fungal Yvclp homologs. We will then test whether the changes in Yvclp's mechanosensitivity under patch clamp match the changes in Ca2+ release in vivo as measured by aequorin luminometry. Yvclp is activated by Ca2+ itself, and we will use mutants to test whether this activation is important in its function in osmotic defense. Others have observed that vacuoles actively shrink and crenellate upon upshock and found that there is a signal-transduction pathway involving phosphoinositide metabolism. Part of our effort will be to integrate our finding on Yvclp with this shrinkage and crenellation. We will examine whether and how these physical changes relate to the function of Yvclp. We will also improve on the techniques to study Yvclp and osmotic responses, including the possible production of Yvclp in frog oocytes or bacteria, rapid perfusion, and the use """"""""cameleon"""""""", as a single-cell Ca2+ reporter.
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