Genetic characterization of the secretory mutants ofParamecium caudatum

Abstract

Two trichocyst-nondischarge (TND) mutants ofParamecium caudatum, tndl andtnd2, are unable to discharge the trichocyst matrix (tmx) in response to chemical stimuli, although they contain many docked trichocysts at predetermined sites in the cortex. Freeze-fracture electron microscopy (FEM) of the plasma membrane showed thattndl possess two typical intramembrane particle arrays at the trichocyst docking site in the cortex, the outer ring and the inner rosette, as observed in wild-type (WT) cells, whereastnd2 possess the ring but not the rosette. The tmx of both TND mutants are able to expand when they are freed and exposed to an extracellular medium containing 1.5 mM Ca2+. When mutant cells were treated with ionophore A23187 and Ca2+, tmx-expansion took place intnd2, but not intndl cells. Thetnd2 mutant could be rescued by an injection of the WT cytoplasm and also by partial cell fusion during conjugation with the WT andtndl cells. However, the secretion capacity oftndl was not restored either by a microinjection of the WT cytoplasm or by the conjugating pair formation. Freeze-fracture electron microscopy on the double homozygote fortndl andtndl genes, revealed only the parenthesis instead of the ring and the rosette, indicating that trichocysts do not dock to the cortical site. Double mutation at thetndl andtndl loci caused a decrease in the number of the trichocysts at the cortical site. These results suggest that cooperative action of the two TND genes is necessary for stable docking of the trichocysts to the cortical sites.