Norbert Klauke

Green fluorescent protein‐tagged sarco(endo)plasmic reticulum Ca2+‐ATPase overexpression in Paramecium cells: isoforms, subcellular localization, biogenesis of cortical calcium stores and functional aspects

We have followed the time‐dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP) attached to the C‐terminus of the PtSERCA1 gene. The outlines of alveolar sacs (ASs) are labelled, as is the endoplasmic reticulum (ER) throughout the cell. When GFP fluorescence is compared with previous anti‐PtSERCA1 antibody labelling, the much wider distribution of GFP (ER+ASs) indicates that only a small amount of SERCA molecules is normally retained in the ER. A second isoform, PtSERCA2, also occurs and its C‐terminal GFP‐tagging results in the same distribution pattern. However, when GFP is inserted in the major cytoplasmic loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, probably because of the presence of the overt C‐terminal KKXX ER‐retention signal and/or masking of a signal for transfer into ASs. On the overall cell surface, new SERCA molecules seem to be permanently delivered from the ER to ASs by vesicle transport, whereas in the fission zone of dividing cells ASs may form anew. In cells overexpressing PtSERCA1 (with C‐terminal GFP) in ASs, [Ca2+]i regulation during exocytosis is not significantly different from controls, probably because their Ca2+ pump has to mediate only slow reuptake.

Caffeine-induced Ca2+ transients and exocytosis in Paramecium cells. A correlated Ca2+ imaging and quenched-flow/freeze-fracture analysis.

Abstract. Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti∼50 to 80 nm, [Ca2+]acti rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent ≥2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge.

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.

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein

Abstract We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and postembedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca2+-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca2+ homeostasis.

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca2+]o (extracellular Ca2+ concentration) is between about 10−4 to 10−3m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+o or increasing [Ca2+]o to 10−2m inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]o) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.