The contractile vacuole of freshwater protozoa is involved in regulation of cell volume and of intracellular solutes (6,13,22) . Despite the longstanding interest in this organelle and knowledge of its functional significance, little is known of the mechanisms of the primary actions of the contractile vacuole, namely, the formation and expulsion of the vacuolar fluid .This report is concerned with the origin of the force for systole, the expulsion of the vacuolar fluid. The force could arise from tension generated in the wall of the vacuole, or from one of several processes in the adjacent cytoplasm, or by a combination of forces . Kitching in 1956 emphasized the lack of pertinent information, but concluded from indirect evidence that the walls of contractile vacuoles develop tension (12) . On the other hand, Wigg et al . (29) suggested that the contractile vacuole (or water-expulsion vesicle) of Amoeba proteus is not contractile, and that the vacuolar fluid is expelled by force generated in the adjacent endoplasm. This conclusion was based on cinemicrography of the vacuolar cycle . The movement of the cytoplasm toward the vacuole during systole and the collapse of the vacuole were taken as evidence for generation of force by the endoplasm, and not by the wall of the vacuole . Similar observations were made on Paramecium (18) and Tetrahymena (17) .We found that the application of adenosine triphosphate (ATP) to contractile vacuoles isolated from Amoeba proteus caused the vacuoles to contract . The contraction had specific requirements for ATP and Mg . This observation suggests that the force for systole is generated by the vacuole itself. A preliminary report of these results has been published (21) .
MATERIALS AND METHODSAmoeba proteus were cultured at 18°C in the dark in a dilute, mixed salt solution (20), and were fed washed Tetrahymena pyriformis .Amoeba proteus normally has one contractile vacuole per cell . The contractile vacuole was identified and distinguished from food vacuoles by its gradually increasing volume (diastole) and by its posterior position in the cell. The vacuole was isolated from the cell with a glass micropipette (outside tip diameter about 30,u) held by hand on the cell just adjacent to the contractile vacuole . A slight, abrupt suction exerted by mouth ruptured the cell plasmalemma and drew the contractile vacuole and a small volume of cytoplasm, free of other vacuoles, into the pipette . The vacuole was placed in a drop of culture medium or other solution in a depression slide . The test solutions applied to the vacuole with micropipettes usually contained ATP or another nucleotide (Sigma Chemical Co ., St . Louis, Mo.), one or more salts, and were adjusted to pH 7.0 . The isolated vacuole was photographed on Polaroid film, before and after treatment, through a microscope equipped with phase-contrast optics .
OBSERVATIONSThe isolated contractile vacuoles were spherical or slightly oval in shape . The diameters were between 20 and 40 µ, and were the same before and for several hours ...