Essentialome-Wide Multigenerational Imaging Reveals Mechanistic Origins of Cell Growth Laws

Eaton, D. S.; Sanchez, C.; Gutierrez-Lopez, L.; Shenker, J. Q.; Goulev, Y.; Watson, B. R.; Henriot, V.; Garner, E. C.; Justman, Q. A.; Moffitt, J. R.; Paulsson, J.

doi:10.1101/2025.06.10.658525

bioRxiv · 10.1101/2025.06.10.658525

Essentialome-Wide Multigenerational Imaging Reveals Mechanistic Origins of Cell Growth Laws

Eaton, D. S.·Sanchez, C.·Gutierrez-Lopez, L.·Shenker, J. Q.·Goulev, Y.·Watson, B. R.·Henriot, V.·Garner, E. C.·Justman, Q. A.·Moffitt, J. R.·Paulsson, J.

View original source ↗Export metadata ↓

Abstract

Escherichia coli is arguably the most thoroughly characterized organism, and yet [~]20% of its essential genes serve completely unknown functions,1,2 and many core quantitative physiological principles remain unexplained, including the famous nutrient growth law where cell volume seems to depend exponentially on growth rate. Here we develop a platform for massive, multi-generational Optical Pooled Screening (OPS)3-6, and apply it to link image-based phenotypes to genotypes for 133,000 CRISPRi knockdowns of essential genes, tracking tens of millions of lineages and analyzing 1.6 billion cells. Our multi-dimensional dynamic phenotypes correlate exceptionally well with known gene functions, allowing us to identify many unknown roles of essential genes. Quantifying the relation between growth and cell size in turn identifies three distinct variants of the bacterial growth laws, which we explain mechanistically by discovering a new role for (p)ppGpp and SpoT as a sensor of translation elongation. Finally, we propose and systematically test an exceedingly simple, passive mechanism for the nutrient growth law, based on triggering cell division through the accumulation of a protein that, unlike ribosomes, is not controlled by (p)ppGpp. The resulting hyperbolic growth law fits the data from our three variants even better than the previously proposed exponential relationship, and with fewer parameters.

Explore related subjects

systems biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Eaton, D. S., Sanchez, C., Gutierrez-Lopez, L., Shenker, J. Q., Goulev, Y., Watson, B. R., Henriot, V., Garner, E. C., Justman, Q. A., Moffitt, J. R., Paulsson, J.. 2025-06-10. Essentialome-Wide Multigenerational Imaging Reveals Mechanistic Origins of Cell Growth Laws. https://doi.org/10.1101/2025.06.10.658525

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Limit-pushing overexpression reveals constraints on protein abundance

Proteins are often classified as toxic or non-toxic without measuring the abundance reached, leaving constraints on tolerable protein abundance unresolved. We established a limit-pushing approach in Saccharomyces cerevisiae combining strong inducible expression with gTOW-mediated high-copy selection to counteract copy-number compensation while measuring protein abundance and growth. Nearly all of approximately 80 chromosome I proteins severely inhibited growth or reduced viability at sufficiently high abundance. We established IE50, the expression level associated with a 50% reduction in growth rate, to quantify their widely varying overexpression tolerance. IE50 was positively associated with predicted structural order and cytoplasmic localization propensity and negatively associated with sulphur content. Single-cell imaging linked higher tolerance to proteins remaining cytoplasmic without becoming aggregation-positive and revealed abundance-dependent changes in localization and organelle morphology. At extreme abundance, Fun12, Nup60, and Pex22 generated distinct large-scale intracellular states through specific sequence regions. These findings establish overexpression toxicity as a quantitative property linked to protein characteristics and reveal both constraints on tolerable abundance and sequence-dependent capacities for intracellular organization.

systems biology↗

bioRxiv2026

Accessing Enzyme Kinetic Data and Prediction Methods at Scale

Enzyme kinetic parameters inform metabolic models, yet experimental measurements are sparse. A growing body of work predicts them from protein and substrate features, but software fragmentation hinders adoption, so downstream tools lock into the most accessible method. We present OpenKinetics Predictor (at predictor.openkinetics.org), an open-source platform integrating thirteen methods in isolated environments behind one interface. The platform optionally reports similarity between query proteins and each method's training data to contextualise reliability. A common featurisation-prediction abstraction keeps it extensible, and independent parties, including original authors, contributed many methods. We pair it with a data portal (at data.openkinetics.org) that exposes CatLog, a curated kinetic dataset, with precomputed embeddings, predicted binding sites, and standardised splits. Both offer a web interface and an API, and the GECKO modelling toolbox already calls the predictor API. As a case study, we predict across an E. coli model and find inter-predictor agreement varies with metabolic context and data availability.

systems biology↗

bioRxiv2026

A thermoregulatory design principle for transitions into hypometabolism

Mammals entering torpor or hibernation undergo an abrupt transition from normothermia to hypothermia, yet how thermoregulation enables this switch remains poorly understood. Here, we identify dynamical signatures that precede these transitions and a mathematical principle that can generate them. In fasting-induced torpor in mice, body-temperature fluctuations increased before torpor onset, providing an early-warning signal that tracked proximity to the transition better than temperature decline alone. A heat-balance model showed that reducing how strongly the effective heat-loss coefficient depends on body temperature reorganizes thermoregulatory stability, allowing a low-temperature equilibrium to emerge while the normothermic state remains stable. This organization is consistent with a symmetry-broken pitchfork involving a saddle-node. Similar increases in temperature fluctuations preceded hibernation onset in hamsters. These findings link pre-transition temperature dynamics to changes in the underlying thermoregulatory landscape and provide a framework for detecting and understanding transitions from normothermia to hypothermia.

systems biology↗