Cellular membranes that undergo cyclic changes in tension: Direct measurement of force generation by an in vitro contractile vacuole of Paramecium multimicronucleatum

The contractile vacuole of the fresh water protozoan Paramecium is a membra ne-bound vesicle that expels excess cytosolic water, acquired osmotically, through its periodic exocytotic activity. The in vitro contractile vacuole, isolated in a small amount of cytosol from the Paramecium cell and confine d under mineral oil, showed periodic rounding and slackening at regular int ervals for an extended time. The contractile vacuole rounded against the cy tosol-mineral oil boundary tension. The tension at the surface of the contr actile vacuole is, therefore, assumed to increase during the rounding phase . We first estimated the tension relative to the boundary tension from the degree of compression of the contractile vacuole by the boundary. We then d etermined the absolute value for the tension at the surface of the contract ile vacuole from the degree of bending of an elastic carbon fiber microcant ilever (8 mum thick; 2 mm long), whose free end was placed at the surface o f an in vitro contractile vacuole. The tension was found to increase to its maximum value of approximately 5 mN m(-1) when the contractile vacuole rou nded, This value was more than 35 times higher than that for the slackened contractile vacuole. Electron micrographs of conventional thin sections of chemically fixed in vitro contractile vacuoles as well as those of in vivo contractile vacuoles obtained from rapid frozen and cryosubstituted cells r evealed the lack of any ultrastructural evidence for the presence of a fibr ous network system surrounding the contractile vacuole. Thus we conclude th at the mechanism(s) by which tension is developed at the surface of the con tractile vacuole membrane resides in the contractile vacuole membrane itsel f. We propose a hypothesis that periodic changes in the spontaneous curvatu re of the contractile vacuole's lipid bilayer membrane is involved in the p eriodic development of higher contractile vacuole membrane tension. The iso lated CV promises to be an excellent model system for understanding the mol ecular mechanisms of the dynamics of biological membrane.