An exocytotic mutant of ParalJ 1 eciulJ 1 caudatulJ 1 : membrane fusion without secretory contents release

This is a detailed characterization of a secretory mutant incapable of releasing secretory contents despite normal exocytotic membrane fusion performance. Trichocyst non-discharge strain tnd1 of Paramecium caudatum and its wildtype (wt) both show a transient cortical [Ca+l; increase and exocytotic membrane fusion in response to the polyamine secretagogue, aminoethyldextran (AEO), or to caffeine, tnd1 cells frequently display spontaneous Ca+ signals parallelled by spontaneous exocytotic membrane fusion. This remains undetected, unless the trichocyst matrix is shown to be freely accessible to the inert, non-membrane permeable fluorochrome, F 2 FITC, from the outside. In these tnd1 cells, spontaneous and AEOor caffeine-induced membrane fusion, always without contents expulsion by decondensation (i.e. several-fold stretching), is ascertained by electron microscopy. Exocytotic openings, with condensed trichocysts retained, may persist for hours without impairing cells. Trichocyst decondensation normally requires micromolar [Ca+]e' but an increase to 10 mM has no effect on tnd1 trichocyst expansion in vivo or in vitro (when isolated and exposed to ionophore A23187 + Ca2+). Paracrystalline packing of the major secretory components (trichynins) does occur, despite incomplete proteolytic precursor processing (according to SOS-PAGE). However, 45Ca+-binding by secretory components is considerably reduced the likely cause of the non-discharge phenotype. Our findings imply significant untriggered membrane fusion in a system normally following the triggered pathway and clear separation of exocytotic membrane fusion from any later Ca+-dependent steps of the secretory cycle.