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 multimicronucleatum exhibits periodic exocytotic activity. This keeps cytosolic osmolarity at a constant value. The contractile vacuole, the central exocytotic vesicle of the complex, becomes disconnected from its surrounding radial arms and rounds before its fluid content is expelled. We previously proposed a hypothesis that the rounding of the contractile vacuole corresponds to an increase in its membrane tension and that a periodic increase in membrane tension governs the exocytotic cycle. We also proposed a hypothesis that transformation of excess planar membrane of the contractile vacuole into 40 nm diameter tubules, that remain continuous with the contractile vacuole membrane, is a primary cause for the tension development in the planar membrane. In order to investigate tension development further, we have examined electron microscopically the contractile vacuole membrane at the rounding 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 vacuole membrane that accompanied the vacuole's rounding was monitored through a fine-tipped microelectrode inserted directly into the in vivo contractile vacuole. A decrease in membrane potential was used to generate an electric signal that activated an injector for injecting a fixative through a microcapillary against the cell at the precise time of rounding. Subsequent electron micrographs of the contractile vacuole membrane clearly demonstrated that numerous approximately 40 nm membrane-bound tubules formed in the vicinity of the vacuole's microtubule ribbons when the vacuole showed rounding. This finding suggested that membrane tubulation was the cause for topographical isolation of excess membrane from the planar membrane during the periodic rounding of the contractile vacuole. This together with stereo-pair images of the contractile vacuole complex membranes suggested that the microtubule ribbons were intimately involved in enhancing this membrane tubulation activity. Electron micrographs of the contractile vacuole complexes also showed that decorated tubules came to lie abnormally close to the contractile vacuole in these impaled cells. This suggested that the contractile vacuole was capable of utilizing the smooth spongiome membrane that lies around the ampullae and the collecting canals to increase its size.
Tominaga
T
T
Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
Naitoh
Y
Y
Allen
R D
RD
D000818:Animals; D003285:Contractile Proteins / Q000502:physiology*; D004594:Electrophysiology; D005089:Exocytosis / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008854:Microscopy, Electron; D018715:Microscopy, Video; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure*; D010247:Paramecium / Q000502:physiology* / Q000648:ultrastructure*; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*; D014617:Vacuoles / Q000502:physiology* / Q000648:ultrastructure*
D003285:Contractile Proteins