Surviving the heat:Heterogeneity of response in Saccharomyces cerevisiae provides insight into thermal damage to the membrane

Environmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains. Using single-cell methods, we demonstrate the extent of heterogeneity of the heat-stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for recovery of entire populations once stresses are removed. The results show that plasma membrane permeability and potential are key factors involved in cell survival, but thermal resistance is not related to homeoviscous adaptation of the plasma membrane. These results have implications for growth and regrowth of populations experiencing environmental heat stress and our understanding of impacts at the level of the single cell. Given the important role of microbes in biofuel production and bioremediation, a thorough understanding of the impact of stress responses of populations and individuals is highly desirable

Surviving the heat:Heterogeneity of response in Saccharomyces cerevisiae provides insight into thermal damage to the membrane

International audienceEnvironmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains.Using single‐cell methods, we demonstrate the extent of heterogeneity of the heat‐stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for recovery of entire populations once stresses are removed. The results show that plasma membrane permeability and potential are key factors involved in cell survival, but thermal resistance is not related to homeoviscous adaptation of the plasma membrane. These results have implications for growth and regrowth of populations experiencing environmental heat stress and our understanding of impacts at the level of the single cell. Given the important role of microbes in biofuel production and bioremediation, a thorough understanding of the impact of stress responses of populations and individuals is highly desirable

Resveratrol-Induced Xenophagy Promotes Intracellular Bacteria Clearance in Intestinal Epithelial Cells and Macrophages

Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be beneficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebrafish model. We also compared the ability of resveratrol derivatives, designed to improve the bioavailability of the parent molecule, to stimulate autophagy and to induce intracellular bacteria clearance. Together, our data demonstrate the ability of resveratrol to stimulate xenophagy, and thereby enhance the clearance of two invasive bacteria involved life-threatening diseases, Salmonella Typhimurium and Crohn's disease-associated Adherent-Invasive Escherichia coli. These findings encourage the further development of pro-autophagic nutrients to strengthen intestinal homeostasis in basal and infectious states

Spectroscopie de Corrélation de Fluorescence : fluidité membranaire et détection de molécule unique en solution concentrée

Fluorescence correlation spectroscopy (FCS) is a single molecule technique very well suitedfor in vivo studies. We have used FCS to explore plasma membrane microfluidity of livingcells. Measurements were conducted at the single cell level, which enabled us to get a detailedoverview of the typical plasma membrane microviscosity distribution of each cell line studied(LR73, MCF7, KB3.1, MESSA and MDCKII). A Monte Carlo simulation based on a 2Ddiffusion model enables us to link the asymetric fluidity distribution profile with the plasmamembrane micro-organization. This result was used to determine the membrane organisationrelated to the surexpression of the P-glycoprotein (Pgp), a protein implicated in multidrugresistance. We also compare the membrane structuration of various cancer cell lines, eachcomes in two versions, a sensitive one and a resistant one to a chemotherapeutic drug : theDoxorubicin.Secondly, we propose a new excitation scheme based on a nonradiative energy transfert.This approach allow us to reduce the illumination depth of the microscope at the nanometricscale. We demonstrate its potential through two applications : FCS in micromolar solutionsand fluorescence imaging on cells adhesion areas.La Spectroscopie de Corrélation de Fluorescence (FCS) est une technique de moléculesuniques particulièrement bien adaptée aux études en milieu biologique. Elle est ici mise enoeuvre pour analyser localement la membrane plasmique de cellules vivantes. Dans un premiertemps, la distribution de la microfluidité membranaire de cellules vivantes a été caractériséegrâce à des mesures de temps de diffusion par FCS. Nous avons ainsi pu établir la loi dedistribution de la fluidité membranaire à l’échelle de la cellule unique pour différentes lignéescellulaires (LR73, MCF7, KB3.1, MESSA et MDCKII). Une simulation Monte-Carlo montrequ’un modèle simple de diffusion à deux dimensions dans une matrice contenant des microdomainesvisqueux peut rendre compte de l’allure asymétrique typique des distributions obtenues.Ce résultat a été utilisé pour évaluer les modifications membranaires liées à la surexpressionde la P-glycoprotéine, protéine responsable d’un phénomène de résistance à la chimiothérapie.Nous avons également comparé la structuration membranaire de diverses lignées cancéreuses,chacune se déclinant en une version sensible et une version résistante à un médicament : laDoxorubicine.Dans un second temps, nous proposons une nouvelle méthode d’illumination locale baséesur un transfert d’énergie non radiatif. Cette méthode permet de réduire la profondeur dechamp du microscope à l’échelle nanométrique. Nous en démontrons ici le potentiel pourl’utilisation de la FCS dans des solutions micromolaires ainsi que pour l’imagerie de l’adhésioncellulaire

Multiphoton Cascade Absorption in Single Molecule Fluorescence Saturation Spectroscopy

International audienceSaturation spectroscopy is a relevant method to investigate photophysical parameters of single fluorescent molecules. Nevertheless, the impact of a gradual increase, over a broad range, of the laser excitation on the intramolecular dynamics is not completely understood, particularly concerning their fluorescence emission (the so-called brightness). Thus, we propose a comprehensive theoretical and experimental study to interpret the unexpected evolution of the brightness with the laser power taking into account the cascade absorption of two and three photons. Furthermore, we highlight the key role played by the confocal observation volume in fluorescence saturation spectroscopy of single molecules in solution

Spectroscopie de corrélation de fluorescence (fluidité membranaire et détection de molécule unique en solution concentrée)

La Spectroscopie de Corrélation de Fluorescence (FCS) est une technique de molécules uniques particulièrement bien adaptée aux études en milieu biologique. Elle est ici mise en œuvre pour analyser localement la membrane plasmique de cellules vivantes. Dans un premier temps, la distribution de la microfluidité membranaire de cellules vivantes a été caractérisée grâce à des mesures de temps de diffusion par FCS. Nous avons ainsi pu établir la loi de distribution de la fluidité membranaire à l échelle de la cellule unique pour différentes lignées cellulaires (LR73, MCF7, KB3.1, MESSA et MDCKII). Une simulation Monte-Carlo montre qu un modèle simple de diffusion à deux dimensions dans une matrice contenant des microdomaines visqueux peut rendre compte de l allure asymétrique typique des distributions obtenues. Ce résultat a été utilisé pour évaluer les modifications membranaires liées à la surexpression de la P-glycoprotéine, protéine responsable d'un phénomène de résistance à la chimiothérapie. Nous avons également comparé la structuration membranaire de diverses lignées cancéreuses, chacune se déclinant en une version sensible et une version résistante à un médicament : la Doxorubicine. Dans un second temps, nous proposons une nouvelle méthode d illumination locale basée sur un transfert d énergie non radiatif. Cette méthode permet de réduire la profondeur de champ du microscope à l échelle nanométrique. Nous en démontrons ici le potentiel pour l'utilisation de la FCS dans des solutions micromolaires ainsi que pour l'imagerie de l'adhésion cellulaireFluorescence correlation spectroscopy (FCS) is a single molecule technique very well suited for in vivo studies. We have used FCS to explore plasma membrane microfluidity of living cells. Measurements were conducted at the single cell level, which enabled us to get a detailed over-view of the typical plasma membrane microviscosity distribution of each cell line studied (LR73, MCF7, KB3.1, MESSA and MDCKII). A Monte Carlo simulation based on a 2D diffusion model enables us to link the asymetric fluidity distribution profile with the plasma membrane micro-organization. This result was used to determine the membrane organisation related to the surexpression of the P-glycoprotein (Pgp), a protein implicated in multidrug resistance. We also compare the membrane structuration of various cancer cell lines, each comes in two versions, a sensitive one and a resistant one to a chemotherapeutic drug: the Doxorubicin. Secondly, we propose a new excitation scheme based on a nonradiative energy transfert. This approach allow us to reduce the illumination depth of the microscope at the nanometric scale. We demonstrate its potential through two applications: FCS in micromolar solutions and fluorescence imaging on cells adhesion areasTROYES-SCD-UTT (103872102) / SudocSudocFranceF

Multiphoton Cascade Absorption in Single Molecule Fluorescence Saturation Spectroscopy

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Multi-photon cascade absorption in single molecule fluorescence saturation spectroscopy

International audienc

Nonradiative Excitation Fluorescence Correlation Spectroscopy

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Shedding Light on the Formation and Structure of Kombucha Biofilm Using Two-Photon Fluorescence Microscopy

International audienceKombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in the de novo formation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected to be the microbiologically active site for cellulose production and spatial optimization of yeast–AAB metabolic interactions. The pellicles then grow in thickness while expanding their layered organization. A comparison with pellicles grown from pure AAB cultures shows differences in consistency and structure that highlight the impact of yeasts on the structure and properties of kombucha pellicles