9603675 Luykx Technical The function of the contractile vacuole will be analyzed through the study of Chlamydomonas mutants. Although contractile vacuole function(s) have been studied in Chlamydomonas and other biological systems from a number of approaches, such as electron microscopy, a systematic genetic approach has not hitherto been used. The Principal Investigator has already isolated several osmoregulatory mutants by means of UV mutagenesis and insertional (plasmid) mutagenesis. The research in this proposal aims to: (1), characterize the mutants genetically and morphologically (by video- and electron microscopy); (2), isolate additional mutants with impaired osmoregulatory function; (3), employ the mutants to define basic processes of the contractile vacuole cycle; and (4) isolate genes regulating water transport and associated membrane fusion events. The information obtained from this work will be helpful in understanding these important and widespread membrane processes in eukaryotic cells generally. Non-Technical Water is required to maintain the living state of cells. All cells regulate the amount of water contained within them by regulating the rate of water exchange with the external environment. Thus, control of cell water content is an important phenomenon in living cells, yet it remains poorly understood. In the unicellular alga Chlamydomonas, as in many other unicellular organisms, cell water content is controlled by the contractile vacuole system, which entails a system of membrane permeability, membrane transport, and membrane fusion. The function of the contractile vacuole involves the segregation and expulsion of water that enters the cell continuously by osmosis. Although it has been studied to some extent by electron microscopy, it has not been subjected to experimental approaches such as genetic analysis. The advantage of using Chlamydomonas for this type of project is that its genetics and cellular biology in general is well described and therefore the contractile vacuole is amenable to efficient investigation. This project will focus on characterizing mutants of the contractile vacuole, which will lead to identification of genes whose products are involved in the control of water balance. The results of this work should have broad significance in the fundamental aspects of water regulation in living cells. ***
