Biology subjects

Pincus, D.

Publications and source records attributed to Pincus, D..

At least 19 recordsLinked to original sources

bioRxiv2026

A Statistical Approach to Cellular Resource Allocation Models

Understanding how cells regulate growth despite molecular complexity remains a central question in quantitative biology. While thousands of genes respond to environmental perturbations, the population growth rate varies smoothly across conditions, suggesting the existence of simple organizing principles. Here, we show that statistical analysis of mRNA composition across environmental conditions reveals growth tradeoffs across organisms including E. coli and S. pombe. Using partial least squares regression, we identify two opposing gene sectors whose coordinated expression encodes growth rate. A minimal transcription-translation model, constrained by empirical scaling laws of total mRNA and ribosomal fractions, explains this tradeoff as a necessary consequence of the empirical observations. Extending the model to include charged tRNA dynamics reveals distinct regulatory regimes: E. coli operates co-limited by ribosomal mRNA and charged tRNA availability, whereas S. cerevisiae is primarily ribosomal mRNA-limited. Together, these results provide a statistical method to determine key tradeoffs across organisms and offer a framework to interpret organism-specific growth regimes.

cell biology↗

bioRxiv2026

A DNA-barcoded Luria-Delbrück assay resolves mechanisms of adaptation

When populations encounter a harsh new environment, they adapt in many different ways - from spontaneous genetic changes to transient non-genetic plasticity. Distinguishing between mechanisms of adaptation is often difficult, as the key events are rare and hard to observe directly. Here, we introduce HiDenSeq, a Luria-Delbruck assay that uses DNA barcoding and deep sequencing to resolve the Luria-Delbruck distribution - the statistics of adaptation to a new environment - over four orders of magnitude. At this statistical depth, the shape of the distribution encodes the underlying adaptive mechanism. We find that DNA mismatch repair mutants shift the distributions scale without changing its shape, indicating their only effect is as mutator alleles. A pulse of UV mutagenesis, however, adds a second, statistically distinguishable mode atop the spontaneous background, showing that different mechanisms of adaptation can be quantified directly from the distribution. By sampling rare-event statistics with DNA barcodes, HiDenSeq provides a general method for studying mechanisms of adaptation in evolving populations from microbes to cancers.

evolutionary biology↗

bioRxiv2026

Chaperone condensates buffer the heat shock response against pleiotropic inputs

Stress response pathways can be specific, dedicated to one stimulus, or pleiotropic, funneling distinct perturbations into a shared program. The heat shock response (HSR), the Hsf1-driven transcriptional program, has been linked to numerous stresses, suggesting it is pleiotropic. Here we find that high amplitude HSR activation is specific to heat shock in budding yeast. Heat shock activates the HSR through a condensate cascade in which orphan ribosomal proteins condense with Sis1 and Hsp70, titrating these chaperones away from their repressive interactions with Hsf1, triggering Hsf1 condensation with the transcriptional machinery, and activating HSR genes. Other stressors likewise drive Sis1 and Hsp70 condensation, redeploying Sis1 to stimulus-specific subcellular sites. However, chaperone condensation is not sufficient to activate the HSR. Sis1 and Hsp70 condense under conditions in which the HSR remains inactive, and Sis1 depletion increases HSR activation under all conditions except heat shock. These results suggest that chaperone condensates buffer the HSR, setting a threshold for activation and imparting specificity.

cell biology↗

bioRxiv2026

Natural Selection in the Wake of Catastrophe

Living organisms, from bacteria to humans, are more likely to survive if their traits enhance fitness. In populations well adapted to their environmental niches, natural selection proceeds via rarely beneficial mutations. But when a catastrophe wipes out niche diversity, sudden adaptation often follows. Here, we present a data-validated theory of natural selection in the wake of catastrophe and unveil a simple law that emerges during recovery: the mean fitness relaxes inversely with time, with a prefactor proportional to the number of traits coupled to the post-catastrophe environment. We put our approach to test using experimental fitness landscapes measured following antibiotic administration to E. coli. The resulting mean trait adaptation is not described by gradient ascent on a fitness landscape, instead it follows an algorithm known as Levenberg-Marquardt optimization. Near fitness peaks, evolutionary trajectories are biased against greediness -- from an optimization perspective, post-catastrophic selection is optimistic.

evolutionary biology↗

bioRxiv2026

Mitochondrial respiration modulates Hsf1 activation and the heat shock response.

Cells employ a bevy of transcriptional and post-translational stress responses to tolerate the burden of misfolded proteins induced by stress. In particular, the heat shock response facilitates the upregulation of molecular chaperones and protein remodeling factors that mediate proteostasis in response to accumulated misfolded proteins in the nucleus and cytosol. However, in response to stress neurons struggle to induce a canonical heat shock response, highlighting our poor understanding of how neurons maintain proteostasis. Specifically, the ability of post-mitotic respiring cells to regulate the heat shock response in comparison to their rapidly dividing, predominantly glycolytic counterparts has been under-studied. In this study, we employ yeast models that are easily manipulated to generate energy via glycolysis or mitochondrial respiration by changing the carbon source in the media. Using this model, we demonstrate that Hsf1 activity, the heat shock response and proteostasis are impaired in respiring cells. Interestingly, our data show that reduced Hsf1 activity regulates viability of respiring cells, with respiring cells poorly tolerating constitutively activated Hsf1. Finally, we describe alternative post-translational programming of the molecular chaperones Hsp70 and Hsp104 that plausibly enables respiring cells to mediate proteostasis despite a dampened heat shock response. Our findings offer new insights into possible proteostatic strategies employed by cells in different metabolic conditions.

molecular biology↗

bioRxiv2026

Physiological architecture and evolutionary origins of cellular adaptability

The capacity to adapt to complex environments ( adaptability) is a defining property of cells. Yet, its relationship across single-cell physiology, population-level responses, and evolutionary timescales remains unclear. We performed single-cell transcriptional profiling of budding yeast across 20 complex environments before and after long-term selection for increased osmotolerance. In ancestral populations, transcriptional responses organized into a reproducible hierarchy where adaptation to certain cues took precedence over others. This hierarchy mechanistically originated within individual cells through contingent regulation of translation initiation. Evolution for >3,000 generations under osmotic stress increased osmotolerance while reordering this hierarchy, selectively deprioritizing osmotic stress as an organizing axis of adaptation. The derived strain exhibited impaired integration of stress responses, defective translational coordination, and reduced fitness outside the selected condition. Together, these findings illustrate that cellular adaptability is an evolved architecture whose form is set by the history of selection. One Sentence SummaryCells adapt to complex environments via a prioritization hierarchy; this hierarchy is shaped by the frequency and magnitude of environmental fluctuations in their evolutionary history.

cell biology↗

bioRxiv2026

Cellquant: a vibecoder's guide to image analysis

Quantitative fluorescence microscopy is central to modern cell biology, yet extracting reproducible measurements from images remains a bottleneck for biologists without programming experience. Here we present cellquant, a single-script command line pipeline for multi-channel fluorescence images that performs cell segmentation, puncta quantification, colocalization analysis, and spatial proximity measurements. Because the interface is entirely text based, the exact command used to generate any result can be recorded and re-executed. We validate cellquant on two biological systems. In human HCT116 cells, the pipeline quantified arsenite-induced stress granule formation. In budding yeast, simultaneous measurement of nucleolar morphology, colocalization, and spatial proximity across a temperature gradient revealed a coordinated sequence of nucleolar reorganization. Applying PCA and UMAP to the multi-parameter output of cellquant resolved a continuous cell state transition across the temperature gradient, with condensate redistribution and nucleolar morphology defining orthogonal axes. The pipeline produces publication-ready quantification with visual quality control and statistically rigorous replicate analysis. All code, documentation, and example datasets are freely available.

cell biology↗

bioRxiv2025

Organelles harbour pH gradients

Organelle pH is critical to organelle identity and function. Resident proteins that define each organelle modify transiting cargo proteins, with both retention and trafficking between organelles governed by pH-dependent mechanisms. For example, lysosomal enzymes bind mannose-6-phosphate receptors at the higher pH ([~]6.5) of the Golgi and dissociate at the lower pH ([~]5.5) of late endosomes1. Proteins that stray from the endoplasmic reticulum (ER) are captured by KDEL receptors in the acidic Golgi and returned into the neutral ER2,3. This pH-tuned trafficking system compartmentalizes organelle function and prevents mis-localization of critical enzymes4. Dysregulated organelle pH disrupts their function and leads to various diseases. Because protons move rapidly in water, the pH within a single organelle is currently assumed to be spatially uniform5. Here, using a reporter sensitive from pH 5.5 - 10.5 to map a spectrum of organelles at high resolution, we discovered that pH gradients exist within single, large or long organelles such as the ER and mitochondria, and in membrane-less organelles without ion-transporting proteins such as the nucleolus. These new findings upend our understanding of organellar pH, prompting new questions about proton diffusion within the cell, and its potential consequences on organelle function.

cell biology↗

bioRxiv2025

Emergent 3D genome reorganization from the stepwise assembly of transcriptional condensates

Transcriptional condensates are clusters of transcription factors, coactivators, and RNA Pol II associated with high-level gene expression, yet how they assemble and function within the cell remains unclear. Here we show that transcriptional condensates form in a stepwise manner to enable both graded and three-dimensional (3D) gene control in the yeast heat shock response. Molecular dissection revealed a condensate cascade. First, the transcription factor Hsf1 clusters upon partial dissociation from the chaperone Hsp70. Next, the coactivator Mediator partitions following further Hsp70 dissociation and Hsf1 phosphorylation. Finally, Pol II condenses, driving the emergent coalescence of HSR genes. Molecular analysis of a series of Hsf1 mutants revealed graded, rather than switch-like, transcriptional activity. Separation-of-function mutants showed that condensate formation can be decoupled from gene activation. Instead, fully assembled HSR condensates promote adaptive 3D genome reconfiguration, suggesting a role of transcriptional condensates beyond gene activation.

molecular biology↗

bioRxiv2024

Feedback control of the heat shock response by spatiotemporal regulation of Hsp70

Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity.

cell biology↗

bioRxiv2022

Adaptive preservation of orphan ribosomal proteins in chaperone-stirred condensates

Ribosome biogenesis is among the most resource-intensive cellular processes, with ribosomal proteins accounting for up to half of all newly synthesized proteins in eukaryotic cells. During stress, cells shut down ribosome biogenesis in part by halting rRNA synthesis, potentially leading to massive accumulation of aggregation-prone "orphan" ribosomal proteins (oRPs). Here we show that during heat shock in yeast and human cells, oRPs accumulate as reversible condensates at the nucleolar periphery recognized by the Hsp70 co-chaperone Sis1/DnaJB6. oRP condensates are liquid-like in cell-free lysate but solidify upon depletion of Sis1 or inhibition of Hsp70. When cells recover from heat shock, oRP condensates disperse in a Sis1-dependent manner, and their ribosomal protein constituents are incorporated into functional ribosomes in the cytosol, enabling cells to efficiently resume growth. One sentence summaryDuring stress, molecular chaperones preserve "orphan" ribosomal proteins (RPs) - RPs that are not bound to rRNA - in liquid-like condensates, maintaining the RPs in a usable form and enabling cells to efficiently resume growth upon recovery from stress.

cell biology↗

bioRxiv2022

Induction of Sis1 promotes fitness but not feedback in the heat shock response

Previously, we combined modeling and experiments to demonstrate that the heat shock response (HSR) functions as a negative feedback loop in which undefined chaperone clients activate the HSR by sequestering Hsp70, and subsequent induction of Hsp70 deactivates the response (Zheng et al., 2016; Krakowiak et al., 2018). Here, we formally define newly synthesized proteins (NSPs) as a major class of HSR activators and determine the role of Sis1, a co-chaperone of Hsp70, in HSR regulation. We develop and experimentally validate a new mathematical model that incorporates NSPs and Sis1. Unexpectedly, genetic decoupling and pulse-labeling experiments reveal that Sis1 induction promotes fitness during prolonged stress rather than providing negative feedback to the HSR. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

cell biology↗

bioRxiv2021

Inducible transcriptional condensates drive 3D genome reorganization in the heat shock response

Mammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved, capable of stress-inducible gene activation or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor Heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, while an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become mammalian-like: constitutively active without intergenic coalescence. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.

molecular biology↗

bioRxiv2021

ReporterSeq reveals genome-wide determinants of proteasome expression

The ubiquitin-proteasome system (UPS) is critical for cellular and organismal health. To uncover mechanisms regulating the UPS in normal and stress conditions, we systematically probed the genome of the eukaryotic model system Saccharomyces cerevisiae for modulators of the UPS master regulator Rpn4 under basal and stress conditions using the reverse genetic method ReporterSeq. The top UPS regulators were the thioredoxin reductase Trr1 and proteins of the large ribosomal subunit, both of which had no previously known role in UPS regulation. Unlike all known mechanisms for Rpn4 regulation which regulate Rpn4 levels, we found that Trr1 modulates the molecular activity of Rpn4 and does so in response to oxidative stress. Our work illuminates the genetic landscape through which cells regulate the UPS, and provides insight into how cells combat proteotoxicity.

genomics↗

bioRxiv2020

Subcellular localization of the J-protein Sis1 regulates the heat shock response

Cells exposed to heat shock induce a conserved gene expression program - the heat shock response (HSR) - encoding chaperones like Hsp70 and other protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1 - a co-chaperone for Hsp70 - controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the cortical ER. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network. One sentence summaryLocalization of the J-protein Sis1 to a subcellular network of proteostasis factors activates the heat shock response.

cell biology↗

bioRxiv2019

A dataset to explore kinase control of environmental stress responsive transcription

Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR) - a gene expression signature shared across the set of perturbations - in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is a constitutive negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling.

systems biology↗

bioRxiv2018

A risk-reward tradeoff of high ribosome production in proliferating cells

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs), which are required to produce thousands of new ribosomes every minute. Although ribosomes are essential in all cells, disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we modeled these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result immediately in acute loss of proteostasis (protein folding homeostasis). Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes. In response, proteostasis genes are activated by an Hsf1-dependent stress response pathway that is required for recovery from r-protein assembly stress. Importantly, we show that exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. Our results highlight ribosome assembly as a linchpin of cellular homeostasis, representing a key proteostasis vulnerability for rapidly proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic conditions that generate orphan r-proteins.

cell biology↗

bioRxiv2018

Transient Hsp90 suppression promotes a heritable change in protein translation

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a unique role promoting the evolution of new heritable traits. To investigate the role of Hsp90 in modulating protein synthesis, we screened more than 1200 proteins involved in mRNA regulation for physical interactions with Hsp90 in human cells. Among the top hits was CPEB2, which strongly binds Hsp90 via its prion domain, reminiscent of the prion-like regulation of translation of Aplysia CPEB. In a yeast model of CPEB prion-dependent translation regulation, transient inhibition of Hsp90 amplified CPEB2 prion activity and resulted in persistent translation of the CPEB reporter. Remarkably, inhibition of Hsp90 was sufficient to induce a heritable change in protein translation that persisted for 30 generations, even in the absence of exogenous CPEB. Although we identified a variety of perturbations that enhanced translation of the reporter, only Hsp90 inhibition led to persistent activation. Thus, transient loss of Hsp90 function leads to the non-genetic inheritance of a novel translational state. We propose that, in addition to sculpting the conformational landscape of the proteome, Hsp90 promotes phenotypic variation by modulating protein synthesis.

cell biology↗