OSMOREGULATORY MUTANTS THAT AFFECT THE FUNCTION OF THE CONTRACTILE VACUOLE IN CHLAMYDOMONAS-REINHARDTII

Four independent osmoregulatory mutants, osm1, osm3, osm4, and osm7, w ere isolated on the basis of their requirement for growth medium of hi gh osmotic strength. In normal low-osmotic-strength medium, in contras t to wild-type cells, the mutants grow poorly or not at all; in distil led water mutant cells are immobilized and eventually swell and burst. The mutants were examined by ordinary brightfield and phase-contrast microscopy, videomicroscopy, and electron microscopy. The four mutants showed different defects in the contractile vacuole (CV) cycle. Timin g of various stages of the CV cycle showed that osm1 was affected prim arily in the early stage of the cycle when the CV begins to grow, osm3 primarily in midcycle when vacuoles fuse to form the CV proper, osm7 at a late stage of the cycle at docking and fusion of the CV with the plasma membrane, and osm4 during contraction of the CV. At the electro n microscopic level, in dilute medium, mutant cells by comparison with wild-type cells had large autophagosomes, swollen mitochondria, and d ilated ER cisternae. Although electron microscopy showed general abnor malities of the contractile vacuoles consistent with the videomicrosco pic observations of living cells, no obvious vacuole membrane abnormal ities were seen which would explain the mutational defects. The mutati ons help define the separate processes that contribute to the coordina ted CV cycle in Chlamydomonas, and open the way to eventual isolation of some of the genes responsible for CV function.