Hyperosmotic stress leads to reversible dissociation of the proton pump-bearing tubules from the contractile vacuole complex in Paramecium.

To study the effect of hyperosmotic stress on the structure and function of the contractile vacuole complex of Paramecium multimicronucleatum, we employed two different monoclonal antibody markers: one to a decorated spongiome antigen (A4) and a second to an antigen found on all other membranes of the contractile vacuole complex (G4). A hyperosmotic condition was produced by adding sorbitol to the axenic culture medium which induced both dose- and time-dependent decreases in the vacuole's expulsion rate. The addition of 150 mM sorbitol to the medium (making a final osmolarity of 230 mOsmol) was sufficient to completely stop the expulsion of the contractile vacuole. Immunofluorescence demonstrated that the blocking of fluid output was accompanied by the disappearance of most fluorescence labeling from the decorated spongiome (the A4 antigen). Electron microscopy revealed that the disappearance of the labeling was accompanied by the disappearance of the decorated tubules from around the collecting canals. These tubules vesiculate. The other membranes of the contractile vacuole complex remained unaffected which was demonstrated by both electron microscopy and indirect immunolabeling using the mAb against the G4 antigen. These results show that the decorated spongiome is formed from a distinct membrane pool separate from that of the smooth spongiome, collecting canals and the contractile vacuole. Recovery of the decorated spongiome rapidly followed the return of the cell to an isotonic environment and was completed within 3 hours. Decorated tubule recovery paralleled the recovery of the function of the contractile vacuole. Recovery was also observed during continuous hyperosmotic treatment with the reappearance of the contractile vacuole activity starting at 3 hours and stabilizing at around 10 hours of incubation. Functional recovery under these conditions was accompanied by a reappearance of the decorated tubules but the total fluid output was always lower than for cells in an isotonic environment. Thus, cells were shown to be capable of adapting to high hyperosmotic conditions. We conclude that the dissociation and reassociation of the decorated spongiome is an important regulatory feature controlling the activity of the contractile vacuole complex and of intracellular osmoregulation in Paramecium.