Biology subjects

Pelet, S.

Publications and source records attributed to Pelet, S..

8 recordsLinked to original sources

bioRxiv2026

Cellular integration of surface and nutrient signals during the early stage of filamentous growth in Saccharomyces cerevisiae

Budding yeast cells can transition from vegetative to filamentous growth under specific environmental stimuli. Saccharomyces cerevisiae has served as a model to understand this process, which is linked to the virulence of many fungal pathogens. One manifestation of filamentation in S. cerevisiae is the formation of pseudohyphal cells, which are elongated and divide in a polarized manner. Here, we established a live-cell imaging assay to monitor this morphogenetic transition and developed a set of fluorescent expression reporters to probe the activity of various signaling pathways during the first 10 hours of this transition. This approach allowed us to identify the stimulus that activates the filamentous growth Mitogen-Activated Protein Kinase (fgMAPK) pathway, which has long evaded identification. Our results indicate that the fgMAPK cascade senses mechanical cues and becomes activated when cells grow on a solid surface. In addition, we have observed that the stimulation of the transcription factor Gln3 regulated by the TOR pathway in low ammonium condition correlates with the elongation of the cells.

cell biology↗

bioRxiv2024

Basal association of a transcription factor favors early gene expression

Responses to extracellular signals via Mitogen-Activated Protein Kinase (MAPK) pathways control complex transcriptional programs where hundreds of genes are induced at a desired level with a specific timing. Gene expression regulation is largely encoded in the promoter of the gene, which harbors numerous transcription factor binding sites. In the mating MAPK pathway of Saccharomyces cerevisiae, one major transcription factor, Ste12, controls the chronology of gene expression necessary for the fusion of two haploid cells. Because endogenous promoters encode a large diversity of Ste12 binding sites (PRE), we engineered synthetic promoters to decipher the rules that dictate mating gene induction. Conformations of PRE dimers that allow efficient gene expression were identified. The strength of binding of Ste12 to the PRE and the distance of the binding sites to the core promoter modulate the level of induction. The speed of activation is ensured by favoring a basal association of Ste12 by using a strong dimer of PRE located in a nucleosome depleted region. Author SummaryDuring development, cell fate decisions allow pluripotent cells to differentiate into various cell types. This process requires cells to integrate signals from their surroundings to initiate a complex transcriptional program. Budding yeasts can also undergo cell fate decisions. In presence of mating pheromones, haploid yeasts can activate a signaling pathway which can ultimately lead to the fusion of two haploid cells to form a diploid. One transcription factor, Ste12, controls this mating transcriptional program. The promoters of these 200 upregulated genes display a large diversity in the organization of Ste12 binding sites. Therefore, it is challenging to decipher how Ste12 regulates the level and the timing of gene expression. To simplify this problem, we have generated synthetic promoters, where the configuration of Ste12 binding sites on the DNA can be controlled. We have identified which conformations of binding site dimers allow a functional association of the transcription factor. In addition, we have also shown that the basal association of Ste12 to the promoter is important for the fast gene induction. An unfavorable configuration of Ste12 binding sites or the presence of nucleosomes restrict the access of the transcription factor to the DNA and results in a slower expression.

cell biology↗

bioRxiv2023

Implication of polymerase recycling for nascent transcript quantification by live cell imaging

Transcription enables the production of RNA from a DNA template. Due to the highly dynamic nature of transcription, live-cell imaging methods play a crucial role in measuring the kinetics of this process. For instance, transcriptional bursts have been visualized using fluorescent phage-coat proteins that associate tightly with mRNA stem loops formed on nascent transcripts. To convert the signal emanating from a transcription site into meaningful estimates of transcription dynamics, the influence of various parameters on the measured signal must be evaluated. Here, the effect of gene length on the intensity of the transcription site focus was analyzed. Intuitively, a longer gene can support a larger number of transcribing polymerases, thus leading to an increase in the measured signal. However, measurements of transcription induced by hyper-osmotic stress responsive promoters display independence from gene length. A mathematical model of the stress-induced transcription process suggests that the formation of gene loops that favor the recycling of polymerase from the terminator to the promoter can explain the observed behavior. One experimentally validated prediction from this model is that the amount of mRNA produced from a short gene should be higher than for a long one as the density of active polymerase on the short gene will be increased by polymerase recycling. Our data suggest that this recycling contributes significantly to the expression output from a gene and that polymerase recycling is modulated by the promoter identity and the cellular state. Take away- Quantification of stress-induced promoter transcription dynamics using a live assays reporter system displays no dependence of signal intensity with gene length. - Mathematical modeling predicts that the formation of gene loops leading to the recycling of polymerases can explain the observed behavior. - More prevalent polymerase recycling on short genes results in a higher transcriptional output.

biochemistry↗

bioRxiv2019

Visualizing cellular heterogeneity by quantifying the dynamics of MAPK activity in live mammalian cells with synthetic fluorescent biosensors

Mitogen-Activated Protein Kinases (MAPKs) control a wide array of cellular functions by transducing extracellular information into defined biological responses. In order to understand how these pathways are regulated, dynamic single cell measurements are highly needed. Fluorescence microscopy is well suited to perform these measurements, however, more dynamic and sensitive biosensors that allow the quantification of signaling activity in living mammalian cells are required. We have engineered a synthetic fluorescent substrate for human MAPKs that relocates from the nucleus to the cytoplasm when phosphorylated by the kinase. We demonstrate that this reporter provides a better sensitivity relative to other similar biosensors and has allowed the monitoring of ERK MAPK activity pulses upon a single physiological EGF stimulation. In addition, we display its applicability to other MAPKs and in multiple cancer cell lines or primary cells as well as its application in vivo in developing tumors. Using our newly developed biosensors, dynamic single cell measurements with high temporal resolution can be obtained. These reporters can be widely applied to the analysis of molecular mechanisms of MAPK signaling in healthy and diseased state, in cell culture assays or in vivo.

cell biology↗

bioRxiv2019

Dynamics of transcription induced by osmostress promoters reveled by live single-cell assays

Precise regulation of gene expression in response to environmental changes is crucial for cell survival, adaptation and proliferation. In eukaryotic cells, extracellular signal integration is often carried out by Mitogen-Activated Protein Kinases (MAPK). Despite a robust MAPK signaling activity, downstream gene expression can display a great variability between single cells. Using a live mRNA reporter, we monitored the dynamics of transcription in Saccharomyces cerevisiae upon hyper-osmotic shock. The transient activity of the MAPK Hog1 opens a temporal window where stress-response genes can be activated. Here we show that the first minutes of Hog1 activity are essential to control the activation of a promoter. The chromatin repression on a locus slows down this transition and contributes to the variability in gene expression, while binding of transcription factors increases the level of transcription. However, soon after Hog1 activity peaks, negative regulators promote chromatin closure of the locus and transcription progressively stops.

biochemistry↗

bioRxiv2019

Close-Range Interactions Favor Growth in Random-Paired Extracted Soil Bacteria

Species interactions at the cellular level are thought to govern the formation and functioning of microbial communities, but direct measurements of species interactions are difficult to perform between the hundreds of different species that constitute most microbial ecosystems. We developed a methodology to examine interactive growth of random cell pairs encapsulated inside 40-70 {micro}m diameter agarose beads. We focused on a sandy soil as a test microbial ecosystem. By using gentle washing procedures, we detached microbial cells from sand and encapsulated them either in the absence or presence of pure culture inoculants. We then tested whether inoculants had on average positive or negative effects on the growth of resident community members depending on the growth substrate. Surprisingly, all the tested inoculants (including Pseudomonas veronii 1YdBTEX2, Pseudomonas putida F1, Pseudomonas protegens CHA0 and Escherichia coli MG1655) stimulated the growth of 40-80 percent of sand-derived cells when grown pair-wise in close proximity (i.e., within the same bead). This was true essentially irrespective of the growth substrate. Beneficial inoculant-sand cell partnerships resulted in up to 100-fold increase in productivity of the sand cell partner and up to 100-fold decrease in that of the inoculant. However, the maximum productivity attained by inoculant-sand cell partners within beads did not surpass that of inoculants alone. Further surprisingly, random pairs of sand cells encapsulated within the same bead also benefited growth in comparison to individual sand cells in a mutualistic manner (i.e., productivity when grown together was greater than the sum of individual productivities), but less than productivities observed in partnerships with the tested inoculants. This suggests that partnerships between inoculants and sand cells are not so much characterized by competition for substrate as by carbon loss through metabolite provision of the inoculant to sand cells (competitive exploitation).

microbiology↗

bioRxiv2018

Cross-regulation between CDK and MAPK control cellular fate

Commitment to a new cell cycle is controlled by a number of cellular signals. Mitogen-Activated Protein Kinase pathways, which transduce multiple extracellular cues, have been shown to be interconnected with the cell cycle. Using budding yeast as a model system, we have quantified in hundreds of live single cells the interplay between the MAPK regulating the mating response and the Cyclin-Dependent Kinase controlling cell cycle progression. Different patterns of MAPK activity dynamics could be identified by clustering cells based on their CDK activity, denoting the tight relationship between these two cellular signals. In mating mixtures, we have verified that the interplay between CDK and MAPK activities allows cells to select their fate, preventing them from being blocked in an undesirable cellular program.

cell biology↗

bioRxiv2017

Timing of gene expression in a cell-fate decision system

During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. Using a dynamic expression reporter, we quantified the kinetics of gene expression in single cells upon exogenous pheromone stimulation and in the physiological context of mating. In both conditions, we observed striking differences in the timing of induction of mating-responsive promoters. Biochemical analyses and generation of synthetic promoter variants demonstrated how the interplay between transcription factor binding and nucleosomes contribute to determine the kinetics of transcription in a simplified cell-fate decision system.\n\nOne Sentence SummaryQuantitative and dynamic single cell measurements in the yeast mating pathway uncover a complex temporal orchestration of gene expression events.

systems biology↗