An exocytotic mutant of Paramecium caudatum: 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 [Ca2+]i increase and exocytotic membrane fusion in response to the polyamine secretagogue, aminoethyldextran (AED), or to caffeine. tnd1 cells frequently display spontaneous Ca2+ 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, F2FITC, from the outside. In these tnd1 cells, spontaneous and AED- or 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 [Ca2+]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 SDS-PAGE). However, 45Ca(2+)-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(2+)-dependent steps of the secretory cycle.
Klauke
N
N
Department of Biology, University of Konstanz, Germany.
Kissmehl
R
R
Plattner
H
H
Haga
N
N
Watanabe
T
T
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D003911:Dextrans / Q000494:pharmacology; D005089:Exocytosis / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects; D008854:Microscopy, Electron; D010247:Paramecium / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D015800:Protozoan Proteins / Q000378:metabolism
D003911:Dextrans; D005456:Fluorescent Dyes; D007476:Ionophores; D015800:Protozoan Proteins; D000001:Calcimycin; D002110:Caffeine; C039691:aminoethyldextran; D002118:Calcium