A key function of non-planar membranes and their associated microtubular ribbons in contractile vacuole membrane dynamics is revealed by electrophysiologically controlled fixation of Paramecium

The contractile vacuole complex of the fresh water protozoan Paramecium mul timicronucleatum exhibits periodic exocytotic activity. This keeps cytosoli c osmolarity at a constant value, The contractile vacuole, the central exoc ytotic vesicle of the complex, becomes disconnected from its surrounding ra dial arms and rounds before its fluid content is expelled. We previously pr oposed a hypothesis that the rounding of the contractile vacuole correspond s to an increase in its membrane tension and that a periodic increase in me mbrane tension governs the exocytotic cycle. We also proposed a hypothesis that transformation of excess planar membrane of the contractile vacuole in to 40 nm diameter tubules, that remain continuous with the contractile vacu ole membrane, is a primary cause for the tension development in the planar membrane. In order to investigate tension development further, we have exam ined electron microscopically the contractile vacuole membrane at the round ing phase. To do this, we developed a computer-aided system to fix the cell precisely at the time that the contractile vacuole exhibited rounding. In this system a decrease in the electrical potential across the contractile v acuole membrane that accompanied the vacuole's rounding was monitored throu gh a fine-tipped microelectrode inserted directly into the in vivo contract ile vacuole, A decrease in membrane potential was used to generate an elect ric signal that activated an injector for injecting a fixative through a mi crocapillary against the cell at the precise time of rounding. Subsequent e lectron micrographs of the contractile vacuole membrane clearly demonstrate d that numerous similar to 40 nm membrane-bound tubules formed in the vicin ity of the vacuole's microtubule ribbons when the vacuole showed rounding, This finding suggested that membrane tubulation was the cause for topograph ical isolation of excess membrane from the planar membrane during the perio dic rounding of the contractile vacuole, This together with stereo-pair ima ges of the contractile vacuole complex membranes suggested that the microtu bule ribbons were intimately involved in enhancing this membrane tubulation activity, Electron micrographs of the contractile vacuole complexes also s howed that decorated tubules came to lie abnormally close to the contractil e vacuole in these impaled cells. This suggested that the contractile vacuo le was capable of utilizing the smooth spongiome membrane that lies around the ampullae and the collecting canals to increase its size.