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 functi on of the contractile vacuole complex of Paramecium multimicronucleatu m, we employed two different monoclonal antibody markers: one to a dec orated spongiome antigen (A4) and a second to an antigen found on all other membranes of the contractile vacuole complex (G4), A hyperosmoti c condition was produced by adding sorbitol to the axenic culture medi um which induced both dose- and time-dependent decreases in the vacuol e's expulsion rate. The addition of 150 mM sorbitol to the medium (mak ing a final osmolarity of 230 mOsmol) was sufficient to completely sto p the expulsion of the contractile vacuole, Immunofluorescence demonst rated that the blocking of fluid output was accompanied by the disappe arance of most fluorescence labeling from the decorated spongiome (the A4 antigen), Electron microscopy revealed that the disappearance of t he labeling was accompanied by the disappearance of the decorated tubu les from around the collecting canals, These tubules vesiculate, The o ther membranes of the contractile vacuole complex remained unaffected which was demonstrated by both electron microscopy and indirect immuno labeling using the mAb against the G4 antigen, These results show that the decorated spongiome is formed from a distinct membrane pool separ ate from that of the smooth spongiome, collecting canals and the contr actile vacuole, Recovery of the decorated spongiome rapidly followed t he return of the cell to an isotonic environment and was completed wit hin 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 contra ctile vacuole activity starting at 3 hours and stabilizing at around 1 0 hours of incubation, Functional recovery under these conditions was accompanied by a reappearance of the decorated tubules but the total f luid output was always lower than for cells in an isotonic environment . Thus, cells were shown to be capable of adapting to high hyperosmoti c conditions, We conclude that the dissociation and reassociation of t he decorated spongiome is an important regulatory feature controlling the activity of the contractile vacuole complex and of intracellular o smoregulation in Paramecium.