Biology subjects

Saez, T.

Publications and source records attributed to Saez, T..

5 recordsLinked to original sources

bioRxiv2025

Mixed positive and negative feedback loops drive diverse single-cell gene expression dynamics

Genetic circuits with only a few components can generate complex gene regulatory dynamics. Here, we combine stochastic modelling and single-cell time-lapse microscopy to reveal the possible dynamics generated by a key gene circuit motif: the mixed positive/negative feedback loop. Our minimal stochastic model of this motif reveals ten distinct classes of dynamic output, including stochastic pulsing, oscillations, and bistability. We systematically map how the circuits core parameters can be tuned to generate each of the behaviours. Experimental validation in two different mixed feedback circuits in the bacterium Bacillus subtilis, {sigma}B and {sigma}V, confirms our models predictive power. Guided by our simulations, we are able to transition between dynamic behaviours by modulating in vivo parameters. Together, these results demonstrate how mixed feedback loops generate diverse single-cell dynamics, improving our understanding of this common biological network motif and informing our efforts to engineer them for synthetic biology applications.

systems biology↗

bioRxiv2024

Environmental and molecular noise buffering by the cyanobacterial clock in individual cells

Circadian clocks enable organisms to anticipate daily cycles, while being robust to molecular and environmental noise. Here, we show how the cyanobacterial clock buffers genetic and environmental perturbations through its core phosphorylation loop. We first characterise single-cell clock dynamics in clock mutants using a microfluidics device that allows precise control of the microenvironment. We find known clock regulators are dispensable for clock robustness, whilst perturbations of the core clock reveal that the wild-type operates at a noise optimum that we can reproduce in a stochastic model of just the core phosphorylation loop. We then examine how the clock responds to noisy environments, including natural light conditions. The model accurately predicts how the clock filters out environmental noise, including fast light fluctuations, to keep time while remaining responsive to environmental shifts. Our findings illustrate how a simple clock network can exhibit complex noise filtering properties, advancing our understanding of how biological circuits can perform accurately in natural environments.

systems biology↗

bioRxiv2023

The alternative sigma factor RpoD4 pulses at division and links the circadian clock and the cell cycle in Synechococcus elongatus

The cyanobacterial circadian clock is an extensive regulator of gene expression, generating a 24-hour rhythm in the majority of genes in Synechococcus elongatus PCC 7942. This raises the question of how the cyanobacterial clock regulates, and receives input from, the diverse cellular processes it controls. Alternative sigma factors, which bind to the core RNA polymerase enzyme and direct the synthesis of specific sets of transcripts, are one mechanism for the clock to set the time of its outputs. In this work, we use single-cell time-lapse microscopy to reveal the transcriptional dynamics of RpoD4, an alternative sigma factor that was previously reported as either arrhythmic or circadian. We find instead that RpoD4 pulses at cell division, dynamics missed by previous bulk averaging. The circadian clock modulates the amplitude of RpoD4 expression pulses, as well as the timing of pulses through its control of division timing. In turn, a rpoD4 mutation causes a reduction in clock period. Further, a rpoD4 mutant results in smaller cell size, and increasing expression of RpoD4 results in larger cell sizes in a dose-response manner, allowing tunable control of cell size. Thus, our single-cell analysis has revealed pulsing gene expression dynamics in cyanobacteria, linking the clock, RpoD4 and the cell cycle.

systems biology↗

bioRxiv2020

Tunable phenotypic variability through an autoregulatory alternative sigma factor circuit

Genetically identical individuals in bacterial populations can display significant phenotypic variability. This variability can be functional, for example by allowing a fraction of stress prepared cells to survive an otherwise lethal stress. The optimal fraction of stress prepared cells depends on environmental conditions. However, how bacterial populations modulate their level of phenotypic variability remains unclear. Here we show that the alternative sigma factor σV circuit in B. subtilis generates functional phenotypic variability that can be tuned by stress level, environmental history, and genetic perturbations. Using single-cell time-lapse microscopy and microfluidics, we find the fraction of cells that immediately activate σV under lysozyme stress depends on stress level and on a memory of previous stress. Iteration between model and experiment reveals that this tunability can be explained by the autoregulatory feedback structure of the sigV operon. As predicted by the model, genetic perturbations to the operon also modulate the response variability. The conserved sigma-anti-sigma autoregulation motif is thus a simple mechanism for bacterial populations to modulate their heterogeneity based on their environment.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2018

WNT ligands stimulate transient signaling in human pluripotent cells and synergize with TGF-β ligands to stimulate sustained signaling during differentiation

WNT/{beta}-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem-cell niches in adults, and is disregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of {beta}-catenin, the primary transducer of canonical WNT signals, using quantitative live-cell imaging. We show that {beta}-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained upon differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell-lines. Signal attenuation in pluripotent cells is controlled by both intra- and extra-cellular negative regulation of WNT signaling. TGF{beta}-superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the {beta}-catenin response in a manner independent of their ability to induce new WNT-ligand production. Our results reveal how variables external to the pathway, including differentiation status and crosstalk with other pathways, dramatically alter WNT/{beta}-catenin dynamics.

developmental biology↗