Contractile vacuoles (CVs), as cellular organelles of protozoa, have been known for 200 years but how they function is only now being discovered. Recent work on Paramecium from this laboratory shows that the CVs contain a hypertonic solution of osmolytes of which K+ and Cl- ions are the most abundant. Being hypertonic to the cytosol, excess water can enter the CV by osmosis from the cytoplasm. It has also been shown that the CV is a unique organelle that undergoes periodic rounding and relaxing cycles, apparently without the aid of a contractile cytoskeletal system. The membrane of the CV contains an innate timing mechanism that triggers CV rounding that is accompanied by a 35- to 50-fold increase in membrane tension. During this rounding excess CV membrane is topographically transformed into 40-nm diameter tubes that remain continuous with the CV. This research will attempt to (1) develop a microcapillary osmometer to measure the total intracellular and intraorganellar osmolarities, (2) determine how K+ and Cl- ions cross the CV membrane into the CV lumen to set up this hypertonic gradient, (3) see how the proton-translocating V-ATPases are used to energize this organelle and (4) determine how the CV can increase its membrane tension so dramatically. To carry on these studies the biochemistry of the CV membrane will be compared with other membranes in Paramecium to determine how this membrane differs from those membranes that are incapable of rounding and tension development. Patch-clamp electrophysiology will than be used to characterize the types of ion channels, water channels and electrogenic pumps present in the CV membranes that can be detected with this technique.
A study of the function of contractile vacuoles, osmoregulatory organelles of protozoa and algae, will be continued. Osmoregulation is the process of balancing water and salts between cells and their environments. Patch clamp electrophysiology and biochemical studies will help us understand how fresh water protozoa have solved the problem of life in an environment where water continuously enters the cell's cytoplasm. Understanding water regulation in "primitive" cells is likely to provide clues on water regulation in much more advanced organisms.
