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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02405

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The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles in Paramecium

¹ Centre National de la Recherche Scientifique, Centre de Génétique Moleculaire, Avenue de la Terasse, Bâtiment 26, F-91198 Gif-sur-Yvette cedex, France
² Universität Konstanz, Fachbereich Biologie, Universitätsstraße 10, 78457 Konstanz, Germany

View larger version (98K): [in a new window]   Fig. 1. Alignment of the c-subunits of P. tetraurelia, S. cerevisiae and M. musculus using the ClustalW algorythm. Residues reported to be involved in bafilomycin binding (Bowman and Bowman, 2002) are printed in red, Glu137 of the yeast c-subunit gene important for proton translocation (Hirata et al., 1997) is printed in yellow.

View larger version (112K): [in a new window]   Fig. 2. Transformation of Paramecium cells with c-subunit-GFP (V0) constructs. Staining with the constructs c1-GFP, c4-GFP and c5-GFP showed no differences; thus, all the c-GFPs seem to be targeted to the same organelles. (a) c5-GFP, strong labeling of the radial canals (rc) of the contractile vacuole complex (cvc); (b) c4-GFP, trichocysts (tr) attached to the cortex are labeled but somewhat obscured by strong cytoplasmic labeling; (c) c4-GFP, the membrane of a food vacuole (fv) is labeled; (d) c4-GFP, when focused on the cell cortex, a regular network is visible, probably representing the ER. Bars, 10 µm.

View larger version (52K): [in a new window]   Fig. 3. GFP localization of the F-subunits of V1. (a) Injection of a moderate amount of F-GFP plasmid into the macronucleus resulted in bright staining of the radial canals (rc) of the contractile vacuole complex; (b) injection of a large amount additionally stained trichocysts (tr), a regular network at the cell cortex, and it caused considerable cytosolic background. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 4. Northern blot of two clones injected with c1-GFP in comparison with non-injected control clones. Overnight exposition of the film allowed the detection of the c1-GFP-mRNA at 1.4 kb whereas the endogenous mRNA of c1 (0.6 kb) is undetectable in transformed and control clones, even after 120 hours of exposure (not shown).

View larger version (34K): [in a new window]   Fig. 5. Application of an anti-GFP antibody to living cells transformed simultaneously with, c1-, c4- and c5-GFP constructs. Food vacuole (fv) membranes are stained whereas the cell surface is devoid of any label; (a) GFP labeling by c-GFP fusion genes; (b) anti-GFP antibody, detected with an Alexa-coupled secondary antibody; (c) untransformed control cell; (d) anti-GFP antibody and secondary antibody applied on a control cell show neither cell-surface labeling nor staining of food vacuole membranes. Bar, 10 µm.

View larger version (114K): [in a new window]   Fig. 6. Silencing of c-subunits of V0 or the F-subunit of V1 causes defects in the cycle of the contractile vacuole complex. (a-d) Control cell, showing normal cycles with filling of the ampullae, fusion with the contractile vacuole and fluid expulsion; (e-h) silencing of any V-ATPase subunit provoked serious perturbation in the series of events and a general prolongation of the cycle, illustrated in this figure by silencing of all c-subunits. Abbreviations: a, ampulla; cv, contractile vacuole; rc, radial canal. Bar, 10 µm.

View larger version (108K): [in a new window]   Fig. 7. EM analysis of the contractile vacuole complex. (a) Control cell compared with one silenced in all c-subunits (b). In cells silenced in any of the c-subunit pairs, the decorated spongiome (cross-sectioned in this figure) is highly disrupted (remaining decorated spongiome is indicated with an arrow) or absent, whereas the smooth spongiome seems to be unaffected. Abbreviations: cc, collecting canal; ds, decorated spongiome; ss, smooth spongiome. Bar, 0.5 µm.

View larger version (12K): [in a new window]   Fig. 8. Silencing of V0- or V1-subunits leads to strongly impaired feeding. Cells 24 hours after the beginning of the silencing experiment were fed for 30 minutes with an E. coli suspension containing a fluorochrome to visualize food vacuoles. After 30 minutes, cells were fixed and the number of food vacuoles of at least 25 cells was counted.

View larger version (14K): [in a new window]   Fig. 9. Comparison of the exocytotic capacity of wild-type paramecia compared with cells silenced in V-ATPase subunits. The exocytotic capacity of c4, c2/c6 and c2/c4/c6 silenced cells is greatly reduced after 24 hours. Exocytotic capacity was analyzed using the picric acid test and three classes of cells were distinguished: `wild-type' with more than 500 expelled trichocysts, `epsilon' with less than 100, and `minus' with no expelled trichocysts. Data were collected in three different experiments from at least 20 cells per group and the variation was calculated between the experiments.

View larger version (129K): [in a new window]   Fig. 10. Cells transformed with trichocyst matrix-protein1b-GFP were silenced in all c-subunits. (a,b,c) control cells show trichocysts docked at the cell cortex at high density; (d,e,f) cells co-silenced in all c-subunits have very few trichocysts attached to the cortex whereas the cytoplasm contains clusters of misshapen trichocyst precursor structures. Bars, 10 µm.

View larger version (113K): [in a new window]   Fig. 11. EM analysis of trichocysts in V0-silenced cells. (a) Control cell showing a trichocyst of normal shape docked to the cell cortex; (b) cells silenced in all c-subunits show various trichocyst misformations, e.g. absence of a proper tip and failure to form the typical spindle-shaped body, thus resembling misshapen trichocysts of the mutant tam38 (Gautier et al., 1994); tr, trichocyst. Bar, 1 µm.

View larger version (111K): [in a new window]   Fig. 12. Cells derived from a clone transformed with F-GFP plasmid were subjected to gene silencing of all the c-subunuits or the F-subunit itself (a-i); 16, 25 and 40 hours after the beginning of feeding, samples were taken and fixed. Control cells (a-c) show no difference in fluorescence within 40 hours, in contrast to cells after silencing of the c-subunits, which causes displacement of the F-subunit from the contractile vacuole complex after 16 hours (d). After 25 hours, only remnants of the contractile vacuole complex are visible and these have disappeared after 40 hours of feeding. Silencing of the F-subunit shows very rapid elimination of the fluorescence of F-GFP already after 16 hours of feeding (g). (j-l) Non-injected control cells showing autofluorescence typical for Paramecium. Silencing of the F-subunit as well as the c-subunits reduces fluorescence of F-GFP to the level of autofluorescence within 40 hours. Bar, 10 µm.

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