9723450 Steck All cells carefully control their water balance to avoid undue swelling and shrinking. While a variety of mechanisms have been implicated, we lack an integrated view of how cytosolic volume is sensed and regulated in eukaryotic cells. An investigation of the molecular basis for water homeostasis in the soil amoeba, Dictyostelium discoideum, is therefore proposed. Of particular interest is the contractile vacuole, a complex organelle which gathers cytosolic water and excretes it from the cell. Although conspicuous to cytologists for the past two centuries, this intracellular nephron is still enigmatic. Dictyostelium discoideum is an ideal organism in which to pursue contractile vacuole complex structure and function. While the full scope of this undertaking is vast, the initial pathway is clear and feasible. A genetic approach has been initiated. Random genes in Dictyostelium were disrupted and vector tags bearing selectable markers inserted into the genome. Mutant strains defective in cell volume homeostasis were selected by visually screening for a swelling phenotype. The marked genes from these lines were rescued as bacterial colonies. The genomic DNA for one such gene, dosA, has been cloned and determination of its sequence is nearly complete. This ~3 kbp open reading frame encodes a putative large, water-soluble protein with multiple sites for potential phosphorylation and interprotein associations zippered by homopolymer stretches. It is now proposed to analyze dosA in detail. The gene copy number will be determined, and the possibility of related forms in a family will be explored. The presence of corresponding genes in other organisms will be examined. The upstream region will be analyzed and compared to other promoter sequences. Putative protein structural features will be predicted by computer analysis. Knock-out mutants (dosA-) will be created and their phenotype explored in detail. Cells will be transformed with dosA constructs tagged at either en d with antigens or GFP protein and the subcellular distribution of the expressed protein will be analyzed by fluorescence microscopy. In addition, antibodies to the epitope tags will be used to search for associated proteins by co-immunoprecipitation. Finally, the structural basis for the function of dosA will be dissected using in vitro mutagenesis and cellular transformation. The relationship of dosA to other water homeostasis genes will be explored as they become identified. Lay abstract: For simple organisms such as protozoa and slime molds (e.g., Dictyostelium discoideum), the maintenance of the proper water balance inside the cell is critical. In severely hypotonic environments (e.g., the soil after a heavy rain), these organisms must be able to pump out the excess water that has entered via passive diffusion across the cell membrane. The specialized organelle that performs this function is known as the "contractile vacuole." Although the role of the cotnractile vacuole and its morphology as determined by microscopy has been known for a long time, it is one of the last subcellular organelles whose mechanism of function remains unknown. This project employs a genetic approach to study the mechanism of contractile vacuole function and water homeostasis in the model organism, Dictyostelium discoideum. It is likely that a better understanding of how water balance is maintained in simple organisms such as D. discoideum will help us understand how cells of higher organisms, including man, regulate water balance and cell volume. ***
