Periodic tension development in the membrane of the in vitro contractile vacuole of Paramecium multimicronucleatum: Modification by bisection, fusionand suction

The contractile vacuole of the freshwater protozoan Paramecium multimicronu cleatum is a membrane-bound exocytotic vesicle that expels excess cytosolic water. The in vitro contractile vacuole isolated from P. multimicronucleat um along with a small amount of cytosol and confined under mineral oil show ed periodic rounding and slackening at fairly regular intervals. Activity l asted for over 30 min at room temperature (24-27 degrees C). The rounding o f the in vitro contractile vacuole corresponded to the increased membrane t ension of the in vivo contractile vacuole that occurs immediately before fl uid expulsion. Unlike the lit vivo contractile vacuole, the in vitro contra ctile vacuole did not expel fluid, since it lacked a mechanism to form a po re. The subsequent slackening of the in vitro contractile vacuole correspon ded to the fluid-filling phase of the in vivo contractile vacuole that occu rs at decreased membrane tension. Fluid filling occurred in the in vivo con tractile vacuole only when it was isolated together with its radial arms. I n vitro membrane-bound vesicles obtained by 'bisecting' (although the two p arts were not always identical in size) an in vitro contractile vacuole est ablished their own independent rounding-slackening cycles. lit vitro contra ctile vacuole vesicles could fuse again when the vesicles slackened. The fu sed vesicle then showed a rounding-slackening cycle with a period closer to that of the vesicle that exhibited the shorter cycle period, An additional rounding phase of the in vitro contractile vacuole could be induced by app lying suction to a portion of its membrane with a micropipette when the con tractile vacuole was in its slackened phase. This suggests that maximum ten sion development in the contractile vacuole membrane can be triggered when tension is increased in any part of the contractile vacuole membrane. The t ime from the start of an extra rounding phase to the next spontaneous round ing and for subsequent rounding-slackening cycles was nearly the same as th at before the extra rounding phase. This implies that there is no master pa cemaker to control the rounding-slackening cycle in the contractile vacuole membrane. Severed radial arms also became vesiculated and, like contractil e vacuole membranes, these in vitro vesicles showed independent rounding-sl ackening cycles and vesicle-vesicle fusions. Thus, membrane derived from th e radial arm seems to be identical in its tension-developing properties wit h the contractile vacuole membrane. ATP was found to be required for contra ctile vacuole rounding but inhibitors of actin or tubulin polymerization, s uch as cytochalasin B and Nocodazole, had no effect on the in vitro contrac tile vacuole's rounding-slackening cycle.