CONTRACTION OF ISOLATED CONTRACTILE VACUOLES FROM AMOEBA PROTEUS

Abstract

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 ...

Functional Characterization of Contractile Vacuole Isolated from Amoeba proteus

“…Moreover, this contraction was very quick. Prusch and Dunham (1970) reported that the CV isolated from A. proteus contracted by 5 mM ATP. However, their results are significantly different from ours.…”

Contractile Vacuoles and Associated Structures: Their Organization and Function

“…This can only be achieved by an active change, on the part of the protozoan cell, in the osmotic equilibrium across its cell membrane. Although it would be possible to achieve this by modifying the permeability of the membrane to water, it appears that there are changes in the cytoplasmic osmotic pressure due to adjustments in the concentration of osmotically active cytoplasmic solutes (Stoner, 1970;Stoner & Dunham, 1970;Prusch, 1977). The time that it takes for this response to lead to a new osmotic equilibrium depends on the organism, on the size of the osmotic shock, and on the specific osmolytes involved.…”

“…Typical compatible solutes are potassium ions, free amino acids, and polyhydric alcohols (Neelon & Bernheim, 1973;Hellebust, 1976;Brown, 1976;Hoffman, 1977 Gagnun (1976, 1978) In addition to the long-term changes typical of cell-volume regulation, short-term changes have been noted in Tetrahymena pyriformis. These take the form of transient but marked changes in sodium and potassium levels (Stoner & Dunham, 1970;Kramherft, 1970;Kropp, 1971 ;Prusch, 1977). At the moment, it is not clear if these changes are active or passive, or to what extent they contribute to the osmoregulatory competence of the cell (see Section IV.…”

“…Riddick (1968) has shown that the sodium concentration of the contractile vacuole fluid is higher than that of the cytoplasm ( Table 2 ) . Further evidence indicates that contractile vacuoles are positively charged with respect to the cytoplasm and this would mean that sodium must be actively transported into them (Yamaguchi, 1960; Josefsson, 1966; Prusch, 1977). All this favours the view that the contractile vacuole complex is involved in the sodium economy of protozoan cells.…”

“…The last two decades have produced major developments in our understanding of the morphological organization of the organelles through electron-microscopical studies, with Rudzinska (1958) and Schneider (1960) setting the early standards, and McKanna (1972McKanna ( , 1973McKanna ( b, 1974McKanna ( , 1976) the later ones. Ion-physiological studies have also influenced our concepts of the functions of contractile vacuoles (Dunham & Child, 1961;Stoner & Dunham, 1970;Dunham & Kropp, 1973;Prusch, 1977).…”