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An attempt to discriminate respiratory dysfunction or mitochondrial uncoupling in yeast mutants by confocal microscopy, image and flow cytometry
274
F L O W A N D I M A G E C Y T O M E T R Y , Montpcllier, D e c e m b e r 1992
J PIIYTOPLANKTONIC SIZE SPECTRUM MODIFICATIONS INDUCED BY SIIELL-FISII IN TIIAU LAGOON (FRANCE). *MAZOUNI Nabila, **COURTIES Claude, *DESLOUS-PAOLI Jean ** Marc, VAQUER Andrd. *IFREMER, Station de S~te, 1, rue J. Vilar, 34207 S~te (France). Lab. d'ltydrobiologie Marine, UM2, URA CNRS 1355, PI. E. Bataillon, 34095 Montpellier Cedex 5 (France). The mediterranean Thau lagoon measuring 75 km2 produces annually 40 000 tons of oysters and nmssels cultured in areas extended for 13.5 km2. Primary producers constitute one of the main feeding source for shell-fish. Phytoplanktonic assemblage, measured by flow cytometry and size fraetionation, is characterized by high concentration of an unknown picophytoplanktonic cells (103-105 ml"1) below 1 p.m and several groups of pltotosynthetie eukaryots (102-104 ml"1) with a sized range from 2 to 30 pm. In situ flow cytometric m.casumments of samples on inside and outside shellfish cultures show a clear decrease in abundance of large cell (>2 p.m) numbers near shell-fish cultures. Experimental assays using oysters in open-flow enclosures corroborate the its situ FCM observations (decrease in >2 ttm size cells). Using batch enclosures it can be shown iha't molluscs arc able to retain smaller cells leading to a dominance of <1 I.tm cells. These results show that if large phytoplanktonic cells arc in low concentrations molluscs are able to filter tim smaller phytoplanktonic cells (0.4 to 0.8 lain). These last features open new questions. Whether or not small cells arc suffisant to ensure the diet ofcuhurcd molluscs ? As molluscs preferentially seem to filter large ceils, are they inducing a selection for the benefit of small cells, particularly concerning the endemic and unknown picoplanktonic population only found in Than lagoon ?
A N A T r E M P T TO DISCRIMINATE R E S P I R A T O R Y D Y S F U N C T I O N O R M I T O C H O N D R I A L U N C O U P L I N G IN YEAST M U T A N T S BY C O N F O C A L MICROSCOPY, IMAGE A N D F L O W C Y T O M E T R Y PETIT" P.X.I, G L A B N. 2,M A R I E D.3, KIEFER H. 4, M E T E Z E A U Ph. 4 and SLONIMSKI p.p.2 IBiosyst~mes Membranaires, CNRS UPR39,Bfit. 24, 2Service de cytomdtrie, ISV, CNRS LP 40 and 3Centre de Gdndtique Moldculaire, CNRS, 91198 Gif.sur-Yvette, France. 4Cytomdtrie Analytique et Prdparative. INSERM-Pasteur-CNRS. Institut Pasteur, 28 rue du Dr. Roux, 75182 Paris, France. Lipophilic cationic cyanine dyes can be used to selectivelystain mitochondria in living yeast in membrane potential-dependent manner. The aim of our study was to extend flow cytometry analysis and sorting to cyanine dye-stained yeast cells and spheroplasts. Experimental conditions were developed that avoid the toxic side effect of the probes (-<0.I ~M) and give a quantitative correlation between fluorescence and mitochondrial membrane potential.The localization of the fluorochrome was checked by confocal microscopy and image cytometry. Differences in light scatter and in fluorescence were detected in mutants (Rho" or Rho °) and wild-type (Rho+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to the wild-type. These characteristics were conserved in spheroplasts produced from the same strains. Application of drugs (i.e.,CCCP, valinomycin) which interferwith the respiratory chain leads to a drastic reduction of the dye uptake. Flow cytometry analysis and sorting allows to selectivelyenrich respiring, non-respiring or drug resistant yeast cells,from the population under study. Confocal microscopy and image cytometry corroborates and confirms the flow cytometry results. Especially concerning the mitochondrial locationof the dyes DiOC6(3) and nonyl acridine orange. They allows to follow the mitochondrial biogenesis during pre-budding and budding states. Quantitative flow cytometry constitute a fast and reproducible technique for rapid screening of yeast strains on which one may suspect respiratory dysfunction or mitochondrial uncoupling following drugs treatment. It is also an adequat method to characterize and isolaterespiratory mutants.
FLOW CYTOMETRY APPROACII OF TIlE PtlYTOPLANKTON STRUCTURE OF THE THAU LAGOON. VAQUER Andrd, COURTII::S Claude Laboratoire d'Hydrobiologie marine, UM II, lIRA CNRS 1355, place E. 8ataillon, 34095 MONTPELLIER Cedex 5 (FRANCE) Phytoplankton community structure of the mediterranean Than lagoon has been studied over a year (1991-1992). Flow eytometry has allowed to describe the population structure by using properties of light scatter and fluorescence induced by cell pigment contents. The algal community is mainly composed o[ centric diatoms, dinoflagcllatcs, eryptophytcs, unspecified phototrophic micro-organisms and unknown picophytoplankton. The size of most of organisms estimated from size fractionation by filtration and from cytometric analysis ranges between 0.4 to 30 p.m. The relative abundance of the different algal sub-populations determines a spatio-temporal phytoplankton structure relatively stable in the lagoon. However in the ostreiculture zone one should note a significant decrease in the cell abundances specially of large size (> 2p.m) under the influence of filter- feeding animals. Flow cytometry has allowed to detect picoalgae (0.4 to 0.Sp.m) which form the major population of the algal assemblage. Cell concentrations vary from 2.103 cells ml-I (in winter) to 1.8.105 cells ml"l (in summer). Picoplankton accounted for 20 to 95% of total algal cell abundance and for 10 to 50% of phototrophic biomass (0.3 to 7 mg m "3 as chlorophyll a) according to seasons and stations. These picoalgae emit only red fluorescence and arc characterized by high chlorophyll b on chlorophyll 11ratio. This lagunar picoplanktonic population is quite different from marine picoplankton observed in areas under influence of Mediterranean Sea. It may be considered as typical of the particular physical and chemical features of lagunar ecosystem and specially of the Thau lagoon. The quantitative significance of this picophytoplankton unknown until now raises questions about the part and the status of this population in the phytoplankton community of the Thau lagoon. As well it raises questions about its contribution to the functioning of the aquatic system (relationships with the different compartments of II~c ecosystem).
M I T O C H O N D R I A L D Y S F U N C T I O N IN YEAST E X P R E S S I N G T H E CYTOPLASMIC MALE S T E R I L I T Y T-urf13 G E N E F R O M MAIZE: AN IMAGE AND F L O W CYTOMETRIC ANALYSIS 1GLA.B N., 2 p E T I T P.X, 3METEZEAU Ph., 3 K I E F E R H. and 1SLONIMSKI P..P. 1Centre de Gdndtique Moldculaire, CNRS U P R 2420, Gif-surYvette, 2Biosyst~mes Membranaires, CNRS UPR 39, Gif-surYvette and 3Cytomdtrie Analytique et Prdparative, INSERMPasteur-CNRS, Institut Pasteur, Paris, France. The u r f 1 3 T W gene which d e r i v e d from the mitechondrial T-urf13 gene responsible for the Texas cytoplasmic male sterility in maize was expressed in Saccharomyces cereuisiae by targeting its translation products into mitochondria (1). Analysis by oxygraphy at population level, reveal that in the presence of methomyl, the oxygen uptake of intact yeast cells carrying the targeted protein is strongly stimulated only with ethanol as respiratory subtrate and not with glycerol, lactate, pyruvate, or acetate. When malate is the substrat oxidized by isolated mitochondria, interaction between the targeted protein and methomyl results in a significant inhibition of the oxygen uptake. This inhibition is canceled and the oxygen uptake is stimulatedby subsequent addition of NAD+. Using a cyanine as probe, interactive laser scanning and flow cytometry, which permit analysis at the cell level, demonstrate that a specific staining of the mitochondrial compartment is obtained and that the cyanine fluorescence is strictly related to the size of the membrane potential (2). Finally, it was shown that as in T cytoplasm maize mitochondria, HInT toxin and methomyl collapse the membrane potential of yeast mitochondria having the foreign protein. Furthermore, results suggest that HInT toxin and methomyl response is related to the plasmid copy number per individual cell and that the deleterious effect induced by the HInT toxin is stronger t h a t this of methomyk (1) GLAB N. et al. (1990) Mol. Gem Genet. 223, 24.32. (2) OLAB N., PETIT P.X. and SLONhMSKIP.P. (1992) biol. Gem Genet. (In press)
F L O W A N D I M A G E C Y T O M E T R Y , Montpcllier, D e c e m b e r 1992
J PIIYTOPLANKTONIC SIZE SPECTRUM MODIFICATIONS INDUCED BY SIIELL-FISII IN TIIAU LAGOON (FRANCE). *MAZOUNI Nabila, **COURTIES Claude, *DESLOUS-PAOLI Jean ** Marc, VAQUER Andrd. *IFREMER, Station de S~te, 1, rue J. Vilar, 34207 S~te (France). Lab. d'ltydrobiologie Marine, UM2, URA CNRS 1355, PI. E. Bataillon, 34095 Montpellier Cedex 5 (France). The mediterranean Thau lagoon measuring 75 km2 produces annually 40 000 tons of oysters and nmssels cultured in areas extended for 13.5 km2. Primary producers constitute one of the main feeding source for shell-fish. Phytoplanktonic assemblage, measured by flow cytometry and size fraetionation, is characterized by high concentration of an unknown picophytoplanktonic cells (103-105 ml"1) below 1 p.m and several groups of pltotosynthetie eukaryots (102-104 ml"1) with a sized range from 2 to 30 pm. In situ flow cytometric m.casumments of samples on inside and outside shellfish cultures show a clear decrease in abundance of large cell (>2 p.m) numbers near shell-fish cultures. Experimental assays using oysters in open-flow enclosures corroborate the its situ FCM observations (decrease in >2 ttm size cells). Using batch enclosures it can be shown iha't molluscs arc able to retain smaller cells leading to a dominance of <1 I.tm cells. These results show that if large phytoplanktonic cells arc in low concentrations molluscs are able to filter tim smaller phytoplanktonic cells (0.4 to 0.8 lain). These last features open new questions. Whether or not small cells arc suffisant to ensure the diet ofcuhurcd molluscs ? As molluscs preferentially seem to filter large ceils, are they inducing a selection for the benefit of small cells, particularly concerning the endemic and unknown picoplanktonic population only found in Than lagoon ?
A N A T r E M P T TO DISCRIMINATE R E S P I R A T O R Y D Y S F U N C T I O N O R M I T O C H O N D R I A L U N C O U P L I N G IN YEAST M U T A N T S BY C O N F O C A L MICROSCOPY, IMAGE A N D F L O W C Y T O M E T R Y PETIT" P.X.I, G L A B N. 2,M A R I E D.3, KIEFER H. 4, M E T E Z E A U Ph. 4 and SLONIMSKI p.p.2 IBiosyst~mes Membranaires, CNRS UPR39,Bfit. 24, 2Service de cytomdtrie, ISV, CNRS LP 40 and 3Centre de Gdndtique Moldculaire, CNRS, 91198 Gif.sur-Yvette, France. 4Cytomdtrie Analytique et Prdparative. INSERM-Pasteur-CNRS. Institut Pasteur, 28 rue du Dr. Roux, 75182 Paris, France. Lipophilic cationic cyanine dyes can be used to selectivelystain mitochondria in living yeast in membrane potential-dependent manner. The aim of our study was to extend flow cytometry analysis and sorting to cyanine dye-stained yeast cells and spheroplasts. Experimental conditions were developed that avoid the toxic side effect of the probes (-<0.I ~M) and give a quantitative correlation between fluorescence and mitochondrial membrane potential.The localization of the fluorochrome was checked by confocal microscopy and image cytometry. Differences in light scatter and in fluorescence were detected in mutants (Rho" or Rho °) and wild-type (Rho+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to the wild-type. These characteristics were conserved in spheroplasts produced from the same strains. Application of drugs (i.e.,CCCP, valinomycin) which interferwith the respiratory chain leads to a drastic reduction of the dye uptake. Flow cytometry analysis and sorting allows to selectivelyenrich respiring, non-respiring or drug resistant yeast cells,from the population under study. Confocal microscopy and image cytometry corroborates and confirms the flow cytometry results. Especially concerning the mitochondrial locationof the dyes DiOC6(3) and nonyl acridine orange. They allows to follow the mitochondrial biogenesis during pre-budding and budding states. Quantitative flow cytometry constitute a fast and reproducible technique for rapid screening of yeast strains on which one may suspect respiratory dysfunction or mitochondrial uncoupling following drugs treatment. It is also an adequat method to characterize and isolaterespiratory mutants.
FLOW CYTOMETRY APPROACII OF TIlE PtlYTOPLANKTON STRUCTURE OF THE THAU LAGOON. VAQUER Andrd, COURTII::S Claude Laboratoire d'Hydrobiologie marine, UM II, lIRA CNRS 1355, place E. 8ataillon, 34095 MONTPELLIER Cedex 5 (FRANCE) Phytoplankton community structure of the mediterranean Than lagoon has been studied over a year (1991-1992). Flow eytometry has allowed to describe the population structure by using properties of light scatter and fluorescence induced by cell pigment contents. The algal community is mainly composed o[ centric diatoms, dinoflagcllatcs, eryptophytcs, unspecified phototrophic micro-organisms and unknown picophytoplankton. The size of most of organisms estimated from size fractionation by filtration and from cytometric analysis ranges between 0.4 to 30 p.m. The relative abundance of the different algal sub-populations determines a spatio-temporal phytoplankton structure relatively stable in the lagoon. However in the ostreiculture zone one should note a significant decrease in the cell abundances specially of large size (> 2p.m) under the influence of filter- feeding animals. Flow cytometry has allowed to detect picoalgae (0.4 to 0.Sp.m) which form the major population of the algal assemblage. Cell concentrations vary from 2.103 cells ml-I (in winter) to 1.8.105 cells ml"l (in summer). Picoplankton accounted for 20 to 95% of total algal cell abundance and for 10 to 50% of phototrophic biomass (0.3 to 7 mg m "3 as chlorophyll a) according to seasons and stations. These picoalgae emit only red fluorescence and arc characterized by high chlorophyll b on chlorophyll 11ratio. This lagunar picoplanktonic population is quite different from marine picoplankton observed in areas under influence of Mediterranean Sea. It may be considered as typical of the particular physical and chemical features of lagunar ecosystem and specially of the Thau lagoon. The quantitative significance of this picophytoplankton unknown until now raises questions about the part and the status of this population in the phytoplankton community of the Thau lagoon. As well it raises questions about its contribution to the functioning of the aquatic system (relationships with the different compartments of II~c ecosystem).
M I T O C H O N D R I A L D Y S F U N C T I O N IN YEAST E X P R E S S I N G T H E CYTOPLASMIC MALE S T E R I L I T Y T-urf13 G E N E F R O M MAIZE: AN IMAGE AND F L O W CYTOMETRIC ANALYSIS 1GLA.B N., 2 p E T I T P.X, 3METEZEAU Ph., 3 K I E F E R H. and 1SLONIMSKI P..P. 1Centre de Gdndtique Moldculaire, CNRS U P R 2420, Gif-surYvette, 2Biosyst~mes Membranaires, CNRS UPR 39, Gif-surYvette and 3Cytomdtrie Analytique et Prdparative, INSERMPasteur-CNRS, Institut Pasteur, Paris, France. The u r f 1 3 T W gene which d e r i v e d from the mitechondrial T-urf13 gene responsible for the Texas cytoplasmic male sterility in maize was expressed in Saccharomyces cereuisiae by targeting its translation products into mitochondria (1). Analysis by oxygraphy at population level, reveal that in the presence of methomyl, the oxygen uptake of intact yeast cells carrying the targeted protein is strongly stimulated only with ethanol as respiratory subtrate and not with glycerol, lactate, pyruvate, or acetate. When malate is the substrat oxidized by isolated mitochondria, interaction between the targeted protein and methomyl results in a significant inhibition of the oxygen uptake. This inhibition is canceled and the oxygen uptake is stimulatedby subsequent addition of NAD+. Using a cyanine as probe, interactive laser scanning and flow cytometry, which permit analysis at the cell level, demonstrate that a specific staining of the mitochondrial compartment is obtained and that the cyanine fluorescence is strictly related to the size of the membrane potential (2). Finally, it was shown that as in T cytoplasm maize mitochondria, HInT toxin and methomyl collapse the membrane potential of yeast mitochondria having the foreign protein. Furthermore, results suggest that HInT toxin and methomyl response is related to the plasmid copy number per individual cell and that the deleterious effect induced by the HInT toxin is stronger t h a t this of methomyk (1) GLAB N. et al. (1990) Mol. Gem Genet. 223, 24.32. (2) OLAB N., PETIT P.X. and SLONhMSKIP.P. (1992) biol. Gem Genet. (In press)