OSMOREGULATION IN PARAMECIUM - THE LOCUS OF FLUID SEGREGATION IN THE CONTRACTILE VACUOLE COMPLEX

In a previous study, monoclonal antibody DS-1 was found to specificall y label the decorated spongiome along the radial arms of the contracti le vacuole complexes in Paramecium multimicronucleatum. Fluorescein is othiocyanate-conjugated DS-1, when injected into cells, labels the rad ial arms initially, but with increasing postinjection time both the in tensity of fluorescence and the number of fluorescently labeled radial arms were reduced. When these cells were fixed after 45 minutes and p robed indirectly using a second fluorochrome, little new label was see n on the already fluorescein-labeled radial arms. Thin sections showed that the amount of decorated tubules along some collecting canals dec reased from the proximal to the distal end and vesicles, which were ne ver seen in control cells, appeared next to the decorated spongiome. T hese results suggested that the decorated spongiome was undergoing dis assembly and sequestration into one region of the cell. The injected D S-1 also reduced the expulsion frequency of the contractile vacuoles i n a dose-, time- and site-dependent manner. The contractile vacuole co mplexes near the injection site were affected more than those farther from the site, but the sizes of both contractile vacuoles were only tr ansiently affected so that fluid output per cell was reduced by approx imately 60%. Beyond 45 minutes postinjection, both the expulsion frequ ency and total fluid output began to recover as the DS-1 was sequester ed into one part of the cell. This region persisted in cells up to 18 hours but disappeared by 24 hours, which coincided with the full recov ery of the expulsion frequency and of decorated spongiome along the ra dial arms. The contractile vacuole, the collecting canals and the smoo th spongiome were morphologically unaffected. These results indicate t hat when the decorated spongiome is dissociated from the contractile v acuole complex, the complex's function is strongly inhibited, showing the decorated spongiome to be the site of fluid segregation.