Mémoire de la réponse au stress chez la levure S. cerevisiae

Cellular memory is a critical ability displayed by micro-organisms in order to adapt to potentially detrimental environmental fluctuations. In the unicellular eukaryote S. cerevisiae, it has been shown at the population level that cellular memory can take the form of a faster or a decreased response following repeated stresses. We here present a study on how yeasts respond to short, pulsed hyperosmotic stresses at the single-cell level. We analyzed the dynamical behavior of the stress responsive STL1 promoter fused to a fluorescent reporter using microfluidics and fluorescence time-lapse microscopy. We established that pSTL1 displays a dynamical variability in its successive activations following two short and repeated stresses. Despite this variability, most cells displayed a memory of past stresses through a decreased activity of pSTL1 upon repeated stresses. We showed that this memory does not require do novo protein synthesis. Rather, the genomic location is important for the memory since promoter displacement to a pericentromeric chromatin domain leads to its decreased transcriptional strength and to the loss of the memory. Interestingly, our results points towards an unreported involvement of the SIR complex on the activity of pSTL1 only when displaced to the pericentromeric domain in our experimental conditions. This study provides a quantitative description of a cellular memory that includes single-cell variability and points towards the contribution of the chromatin structure in stress memory. Our work could serve as a basis to broader studies on the positioning of stress response genes at subtelomeric positions in the budding yeast, from a genetic point of view as well as an evolutionary one.La mémoire cellulaire est une capacité critique dont font preuve les micro-organismes pour s'adapter aux fluctuations environnementales potentiellement néfastes. Chez l'eucaryote unicellulaire S. cerevisiae, il a été montré à l’échelle d’une population que la mémoire cellulaire peut prendre la forme d'une réponse plus rapide ou moins prononcée suite à des stress répétés. Nous présentons ici une étude sur la façon dont les levures réagissent à des stress hyperosmotiques de courte durée à l’échelle de la cellule unique. Nous avons analysé le comportement dynamique du promoteur STL1, exprimé en condition de stress osmotique, et fusionné à un rapporteur fluorescent en faisant usage de microfluidique et de microscopie à fluorescence. Nous avons établi que pSTL1 présente une variabilité dynamique dans ses activations successives après deux stress courts. Malgré cette variabilité, la plupart des cellules présentent une mémoire des stress passés caractérisée par une diminution de l'activité de pSTL1. Nous avons montré que cette mémoire ne nécessite pas de nouvelle synthèse de protéines. L'emplacement génomique est important pour cette mémoire puisque le déplacement du promoteur vers un domaine chromatinien péricentromérique entraîne une diminution de sa force transcriptionnelle ainsi que la perte de la mémoire. Nos résultats indiquent aussi une implication non rapportée du complexe SIR sur l'activité de pSTL1 lorsqu'il est déplacé dans le domaine péricentromérique, dans nos conditions expérimentales. Cette étude fournit une description quantitative d'une mémoire cellulaire qui inclut la variabilité cellulaire et prend en compte la contribution de la structure de la chromatine sur la mémoire du stress. Nos travaux pourraient servir de base à des études plus larges sur le positionnement des gènes de réponse au stress en positions subtélomériques dans la levure, tant d'un point de vue génétique qu'évolutif

Hyperosmotic Stress Response Memory is Modulated by Gene Positioning in Yeast

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Enrichment of persisters enabled by a beta-lactam-induced filamentation method reveals their stochastic single-cell awakening

When exposed to lethal doses of antibiotics, bacterial populations are most often not completely eradicated. A small number of phenotypic variants, defined as 'persisters', are refractory to antibiotics and survive treatment. Despite their involvement in relapsing infections, processes determining phenotypic switches from and to the persister state largely remain elusive. This is mainly due to the low frequency of persisters and the lack of reliable persistence markers, both hampering studies of persistence at the single-cell level. Here we present a highly effective persister enrichment method involving cephalexin, an antibiotic that induces extensive filamentation of susceptible cells. We used our enrichment method to monitor outgrowth of Escherichia coli persisters at the single-cell level, thereby conclusively demonstrating that persister awakening is a stochastic phenomenon. We anticipate that our approach can have far-reaching consequences in the persistence field, by allowing single-cell studies at a much higher throughput than previously reported.status: publishe

Enrichment of persisters enabled by a ß-lactam-induced filamentation method reveals their stochastic single-cell awakening

International audienceAbstract When exposed to lethal doses of antibiotics, bacterial populations are most often not completely eradicated. A small number of phenotypic variants, defined as ‘persisters’, are refractory to antibiotics and survive treatment. Despite their involvement in relapsing infections, processes determining phenotypic switches from and to the persister state largely remain elusive. This is mainly due to the low frequency of persisters and the lack of reliable persistence markers, both hampering studies of persistence at the single-cell level. Here we present a highly effective persister enrichment method involving cephalexin, an antibiotic that induces extensive filamentation of susceptible cells. We used our enrichment method to monitor outgrowth of Escherichia coli persisters at the single-cell level, thereby conclusively demonstrating that persister awakening is a stochastic phenomenon. We anticipate that our approach can have far-reaching consequences in the persistence field, by allowing single-cell studies at a much higher throughput than previously reported

A microfluidic mechano-chemostat for tissues and organisms reveals that confined growth is accompanied with increased macromolecular crowding

International audienceConventional culture conditions are oftentimes insufficient to study tissues, organisms, or 3D multicellularassemblies. They lack both dynamic chemical and mechanical control over the microenvironment. While specific microfluidic devices have been developed to address chemical control, they often do not allow the control of compressive forces emerging when cells proliferate in a confined environment. Here, we present a generic microfluidic device to control both chemical and mechanical compressive forces. This device relies on the use of sliding elements consisting of microfabricated rods that can be inserted inside a microfluidic device. Sliding elements enable the creation of reconfigurable closed culture chambers for the study of whole organisms or model micro-tissues. By confining the micro-tissues, we studied the biophysical impact of growth-induced pressure and showed that this mechanical stress is associated with an increase in macromolecular crowding, shedding light on this understudied type of mechanical stress. Our mechano-chemostat allows the long-term culture of biological samples and can be used to study both the impact of specific conditions as well as the consequences of mechanical compression