Biology subjects

Elston, T. C.

Publications and source records attributed to Elston, T. C..

16 recordsLinked to original sources

bioRxiv2026

RhoG, Rac1 and Cdc42 cooperation in cell protrusion revealed bymultiplexed optogenetics and biosensor imaging

The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoGs effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.

cell biology↗

bioRxiv2026

Quantitative analysis of fibroblast migration reveals migratory states characterized by force generation, cell shape and motion

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

cell biology↗

bioRxiv2026

Transcriptomic profiling of mouse mammary tumors enables prognostic and predictive biomarker discovery for human breast cancers

The development and validation of prognostic and predictive biomarkers in breast cancer is limited by the availability of well-annotated datasets linking tumor molecular features to treatment response and survival outcomes. To address this need, we generated an extensive mouse models dataset comprised of 26 immunocompetent mammary tumor models spanning diverse genetic backgrounds, epithelial-mesenchymal states, the basal-luminal axis, and distinct immune microenvironments. For each model, we measured survival under no treatment, immune checkpoint inhibition (ICI), and carboplatin/paclitaxel chemotherapy. We performed RNA-seq on baseline tumors and on 7-day on-treatment samples for both regimens. Using baseline murine tumor gene expression features, we trained a machine learning Elastic Net model that predicted survival outcomes on multiple human breast cancer datasets with performance comparable to that of existing prognostic assays. We next trained models for ICI benefit, using either the untreated or 7-day ICI treated samples; both models predicted ICI benefit on human ICI treated datasets, with the 7-day treated tumor model showing better performance. We also developed a predictor of carboplatin/paclitaxel response that performed well in mice but did not generalize to human chemotherapy cohorts. Finally, we compared multiple computational approaches, including XGBoost, random forests, and support vector regression; all methods successfully predicted survival outcomes, with Elastic Net offering the best performance and interpretability. These results indicate conserved cancer biology between mouse and human tumors for prognosis and ICI response and establish this large preclinical dataset with linked phenotypic and genomic data, as a resource for benchmarking computational methods for survival prediction. SignificanceThe development of a genomically and phenotypically diverse murine tumor dataset with linked treatment outcomes establishes a robust translational resource to develop, test, and benchmark clinically relevant prognostic and therapeutic response biomarkers.

cancer biology↗

bioRxiv2025

A phase oscillator model of cell cycles reveals nuclear density control in a branched fungal network

Multinucleate cells are widespread in biology, from human skeletal muscle and placenta to many filamentous fungi, yet their basic cell biology remains poorly understood. Maintaining an appropriate nuclear-to-cytoplasmic ratio is essential across cell types for physiological function, and mechanisms of size control have been extensively studied in mononucleate cells. Much less is known about how comparable control is achieved in cells where many nuclei share a common cytoplasm. The filamentous fungus Ashbya gossypii forms a branching mycelial network in which individual nuclei divide asynchronously, while the number of nuclei per cell volume (the nuclear density) is tightly controlled. How global regulation of nuclear density coexists with local cell cycle asynchrony remains unclear. We address this by combining a mathematical model of nuclear division in a growing and branching cell with live-cell microscopy. We model nuclei as a dividing population of phase oscillators within a branching cell network and parameterize the model with measurements from Ashbya cells. The model demonstrates that asynchrony is required to prevent large density fluctuations that would result from synchronous division, and that introducing a nuclear-density checkpoint to the cell cycles leads to synchrony if it is the only mechanism of density control. We find that coupling branch formation to nuclear density both stabilizes nuclear density and prevents the emergence of synchronous cycles. Our results indicate that asynchronous nuclear cycles together with density-responsive branching maintain a constant nuclear density, revealing a strategy for regulating the nuclear-to-cytoplasmic ratio in large multinucleate cells. SignificanceMultinucleate cells appear in diverse biological contexts, from human tissues to filamentous fungi, yet many fundamental aspects of their cell biology are still unclear. Regulating the nuclear-to-cytoplasmic ratio is important across cell types, and little is known about how this is achieved in large, multinucleate fungal cells. Using mathematical modeling and live-cell imaging, we identify how the density of nuclei is controlled in a growing and branching fungal mycelial network. We find that asynchronous nuclear cycles together with density-responsive branching can stabilize the nuclear-to-cytoplasmic ratio within a growing fungal network. This illustrates how large multinucleate cells can control nuclear density even as their morphology becomes increasingly complex.

cell biology↗

bioRxiv2025

Coupling mathematical modeling with a novel human intestinal stem cell system to understand feedback regulation during planar cell polarity

The Planar Cell Polarity (PCP) complex regulates many diverse phenotypes. While recent literature has elucidated key mechanisms underlying PCP, a mechanistic understanding of how these components function as a system to drive polarity is lacking. Here, we develop a comprehensive multicellular mathematical model centered around key PCP phosphorylation events, directly simulating the protein interactions that drive PCP. Our model confirms key PCP phenotypes, including robust single-junction asymmetry and multicellular polarity alignment in the absence of extrinsic signals. It predicts unique roles for the two known positive feedback mechanisms and predicts that VANGL-mediated DVL phosphorylation may be an underappreciated negative feedback mechanism. To test model predictions, we employ transgenic primary human intestinal epithelium cultured on biomimetic planar-crypt microarrays (PCMs) as a new platform for studying PCP. Together, our model provides novel insights into the mechanisms that regulate PCP, while our experimental results highlight an unappreciated role for PCP in intestinal biology.

systems biology↗

bioRxiv2024

Negative feedback equalizes polarity sites in a multi-budding yeast

Morphogenesis in fungi and animals is directed by polarization of small GTPases Cdc42 and Rac. In the budding yeast Saccharomyces cerevisiae competition between polarity patches results in one polarized patch and the growth of a single bud. Here, we describe cell polarity in the yeast Aureobasidium pullulans, which establishes multiple coexisting polarity sites yielding multiple buds during a single cell division cycle. Polarity machinery components oscillate in their abundance in these coexisting sites but do so independently of one another, pointing to a lack of global coupling between sites. Previous theoretical work has demonstrated that negative feedback in a polarity circuit could promote coexistence of multiple polarity sites, and time-delayed negative feedback is known to cause oscillations. We show that both these features of negative feedback depend on a protein we identified as Pak1, and that Pak1 requires Rac1 but not Cdc42 for its localization. This work shows how conserved signaling networks can be modulated for distinct morphogenic programs even within the constraints of fungal budding.

cell biology↗

bioRxiv2023

Particle-based simulations reveal two positive feedback loops allow relocation and stabilization of the polarity site during yeast mating

Many cells adjust the direction of polarized growth or migration in response to external directional cues. The yeast Saccharomyces cerevisiae orient their cell fronts (also called polarity sites) up pheromone gradients in the course of mating. However, the initial polarity site is often not oriented towards the eventual mating partner, and cells relocate the polarity site in an indecisive manner before developing a stable orientation. During this reorientation phase, the polarity site displays erratic assembly-disassembly behavior and moves around the cell cortex. The mechanisms underlying this dynamic behavior remain poorly understood. Particle-based simulations of the core polarity circuit revealed that molecular-level fluctuations are insufficient to overcome the strong positive feedback required for polarization and generate relocating polarity sites. Surprisingly, inclusion of a second pathway that promotes polarity site orientation generated a mobile polarity site with properties similar to those observed experimentally. This pathway forms a second positive feedback loop involving the recruitment of receptors to the cell membrane and couples polarity establishment to gradient sensing. This second positive feedback loop also allows cells to stabilize their polarity site once the site is aligned with the pheromone gradient. Author summaryCells perform many complex tasks, including directed growth, migration, division and differentiation. To accomplish these tasks, the relevant molecular machinery is localized to specific cellular regions. The asymmetric distribution of cellular components is referred to as cell polarity. Polarity is established by localized activation of the protein Cdc42. Establishing mechanisms that regulate the spatiotemporal activity of Cdc42 is a fundamental area of cell biology. Mating yeast cells dynamically relocate a region of high Cdc42 activity, referred to as the polarity site, and grow toward each other after proper alignment of the sites. We investigated mechanisms that generate dynamic polarity sites by performing particle-based simulations of the biochemical reactions that regulate Cdc42 activity. The reactions contain two positive feedback loops that reinforce Cdc42 activity. The first involves autocatalytic activation of Cdc42 through recruitment of an activator. While necessary for polarity establishment, this feedback loop on its own created a stable polarity site that did not relocate. Incorporation of the second feedback loop, which couples the polarity machinery to extracellular mating signals, generated mobile polarity sites. This feedback loop also provides a mechanism for developing stable alignment of polarity sites. Our findings provide insight into how cells regulate polarity dynamics to accomplish complex tasks.

systems biology↗

bioRxiv2022

Actin nano-architecture of phagocytic podosomes

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an "hourglass" shape of the podosome actin core, a protruding "knob" at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, -actinin, cortactin, and microtubules relative to actin were characterized.

cell biology↗

bioRxiv2022

Spatial Models of Pattern Formation During Phagocytosis

Phagocytosis, the biological process in which cells ingest large particles such as bacteria, is a key component of the innate immune response. Fc{gamma} receptor (Fc{gamma}R)-mediated phagocytosis is initiated when these receptors are activated after binding immunoglobulin G (IgG). Receptor activation initiates a signaling cascade that leads to the formation of the phagocytic cup and culminates with ingestion of the foreign particle. In the experimental system termed "frustrated phagocytosis", cells attempt to internalize micropatterned disks of IgG. Cells that engage in frustrated phagocytosis form "rosettes" of actin-enriched structures called podosomes around the IgG disk. The mechanism that generates the rosette pattern is unknown. We present data that supports the involvement of Cdc42, a member of the Rho family of GTPases, in pattern formation. Cdc42 acts downstream of receptor activation, upstream of actin polymerization, and is known to play a role in polarity establishment. Reaction-diffusion models for GTPase spatiotemporal dynamics exist. We demonstrate how the addition of negative feedback and minor changes to these models can generate the experimentally observed rosette pattern of podosomes. We show that this pattern formation can occur through two general mechanisms. In the first mechanism, an intermediate species forms a ring of high activity around the IgG disk, which then promotes rosette organization. The second mechanism does not require initial ring formation but relies on spatial gradients of intermediate chemical species that are selectively activated over the IgG patch. Finally, we analyze the models to suggest experiments to test their validity. Author SummaryPhagocytosis, the process by which cells ingest foreign bodies, plays an important role in innate immunity. Phagocytosis is initiated when antibodies coating the surface of a foreign body are recognized by immune cells, such as macrophages. To study early events in phagocytosis, we used "frustrated phagocytosis", an experimental system in which antibodies are micropatterned in disks on a cover slip. The cytoskeleton of cells attempting to phagocytose these disks organizes into "rosette" patterns around the disks. To investigate mechanisms that underlie rosette formation we turned to mathematical modeling based on reaction-diffusion equations. Building on existing models for polarity establishment, our analysis revealed two mechanisms for rosette formation. In the first scenario an initial ring of an intermediate signaling molecule forms around the disk, while in the second scenario rosette formation is driven by gradients of positive and negative pathway regulators that are activated over the disk. Finally, we analyze our models to suggest experiments for testing these mechanisms.

systems biology↗

bioRxiv2020

A novel stochastic simulation approach enables exploration of mechanisms for regulating polarity site movement

Cells polarize their movement or growth toward external directional cues in many different contexts. For example, budding yeast cells grow toward potential mating partners in response to pheromone gradients. Directed growth is controlled by polarity factors that assemble into clusters at the cell membrane. The clusters assemble, disassemble, and move between different regions of the membrane before eventually forming a stable polarity site directed toward the pheromone source. Pathways that regulate clustering have been identified but the molecular mechanisms that regulate cluster mobility are not well understood. To gain insight into the contribution of chemical noise to cluster behavior we simulated clustering within the reaction-diffusion master equation (RDME) framework to account for molecular-level fluctuations. RDME simulations are a computationally efficient approximation, but their results can diverge from the underlying microscopic dynamics. We implemented novel concentration-dependent rate constants that improved the accuracy of RDME-based simulations of cluster behavior, allowing us to efficiently investigate how cluster dynamics might be regulated. Molecular noise was effective in relocating clusters when the clusters contained low numbers of limiting polarity factors, and when Cdc42, the central polarity regulator, exhibited short dwell times at the polarity site. Cluster stabilization occurred when abundances or binding rates were altered to either lengthen dwell times or increase the number of polarity molecules in the cluster. We validated key results using full 3D particle-based simulations. Understanding the mechanisms cells use to regulate the dynamics of polarity clusters should provide insights into how cells dynamically track external directional cues. Author summaryCells localize polarity molecules in a small region of the plasma membrane forming a polarity cluster that directs functions such as migration, reproduction, and growth. Guided by external signals, these clusters move across the membrane allowing cells to reorient growth or motion. The polarity molecules continuously and randomly shuttle between the cluster and the cell cytosol and, as a result, the number and distribution of molecules at the cluster constantly changes. Here we present an improved stochastic simulation algorithm to investigate how such molecular-scale fluctuations induce cluster movement across the cell membrane. Unexpectedly, cluster mobility does not correlate with variations in total molecule abundance within the cluster, but rather with changes in the spatial distribution of molecules that form the cluster. Cluster motion is faster when polarity molecules are scarce and when they shuttle rapidly between the cluster and the cytosol. Our results suggest that cells control cluster mobility by regulating the abundance of polarity molecules and biochemical reactions that affect the time molecules spend at the cluster. We provide insights into how cells harness random molecular behavior to perform functions important for survival, such as detecting the direction of external signals.

systems biology↗

bioRxiv2020

A predictive model of gene expression reveals the role of regulatory motifs in the mating response of yeast

ABSTRACTCells use signaling pathways to receive and process information about their environment. These systems are nonlinear, relying on feedback and feedforward regulation to respond appropriately to changing environmental conditions. Mathematical models developed to describe signaling pathways often fail to show predictive power, because the models are not trained on data that probe the diverse time scales on which feedforward and feedback regulation operate. We addressed this limitation using microfluidics to expose cells to a broad range of dynamic environmental conditions. In particular, we focus on the well-characterized mating response pathway of S. cerevisiae (yeast). This pathway is activated by mating pheromone and initiates the transcriptional changes required for mating. Although much is known about the molecular components of the mating response pathway, less is known about how these components function as a dynamical system. Our experimental data revealed that pheromone-induced transcription persists following removal of pheromone and that long-term adaptation of the transcriptional response occurs when pheromone exposure is sustained. We developed a model of the regulatory network that captured both persistence and long-term adaptation of the mating response. We fit this model to experimental data using an evolutionary algorithm and used the parameterized model to predict scenarios for which it was not trained, including different temporal stimulus profiles and genetic perturbations to pathway components. Our model allowed us to establish the role of four regulatory motifs in coordinating pathway response to persistent and dynamic stimulation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2020

Molecular switch architecture drives response properties

Many intracellular signaling pathways are composed of molecular switches, proteins that transition between two states--on and off. Typically, signaling is initiated when an external stimulus activates its cognate receptor that in turn causes downstream switches to transition from off to on using one of the following mechanisms: activation, in which the transition rate from the off state to the on state increases; derepression, in which the transition rate from the on state to the off state decreases; and concerted, in which activation and derepression operate simultaneously. We use mathematical modeling to compare these signaling mechanisms in terms of their dose-response curves, response times, and abilities to process upstream fluctuations. Our analysis elucidates several general principles. First, activation increases the sensitivity of the pathway, whereas derepression decreases sensitivity. Second, activation generates response times that decrease with signal strength, whereas derepression causes response times to increase with signal strength. These opposing features allow the concerted mechanism to not only show dose-response alignment, but also to decouple the response time from stimulus strength. However, these potentially beneficial properties come at the expense of increased susceptibility to up-stream fluctuations. In addition to above response metrics, we also examine the effect of receptor removal on switches governed by activation and derepression. We find that if inactive (active) receptors are preferentially removed then activation (derepression) exhibits a sustained response whereas derepression (activation) adapts. In total, we show how the architecture of molecular switches govern their response properties. We also discuss the biological implications of our findings.

systems biology↗

bioRxiv2020

Model-driven experimental design identifies counter-acting feedback regulation in the osmotic stress response of yeast

Cells rely on mitogen-activated protein kinases (MAPKs) to survive environmental stress. In yeast, activation of the MAPK Hog1 is known to mediate the response to high osmotic conditions. Recent studies of Hog1 revealed that its temporal activity is subject to both negative and positive feedback regulation, yet the mechanisms of feedback remain unclear. By designing mathematical models of increasing complexity for the Hog1 MAPK cascade, we identified pathway circuitry sufficient to capture Hog1 dynamics observed in vivo. We used these models to optimize experimental designs for distinguishing potential feedback loops. Performing experiments based on these models revealed mutual inhibition between Hog1 and its phosphatases as the likely positive feedback mechanism underlying switch-like, dose-dependent MAPK activation. Importantly, our findings reveal a new signaling function for MAPK phosphatases. More broadly, they demonstrate the value using mathematical models to infer targets of feedback regulation in signaling pathways.

systems biology↗

bioRxiv2019

Deep learning enables structured illumination microscopy with low light levels and enhanced speed

Using deep learning to augment structured illumination microscopy (SIM), we obtained a fivefold reduction in the number of raw images required for super-resolution SIM, and generated images under extreme low light conditions (100X fewer photons). We validated the performance of deep neural networks on different cellular structures and achieved multi-color, live-cell super-resolution imaging with greatly reduced photobleaching.

biophysics↗

bioRxiv2018

Stochastic methods for inferring states of cell migration

Cell migration refers to the ability of cells to translocate across a substrate or through a matrix. To achieve net movement requires spatiotemporal regulation of the actin cytoskeleton. Computational approaches are neceary to identify and quantify the regulatory mechanisms that generate directed cell movement. To address this need, we developed computational tools, based on stochastic modeling, to analyze time series data for the position of randomly migrating cells. Our approach allows parameters that characterize cell movement to be efficiently estimated from time series data. We applied our methods to analyze the random migration of Mouse Embryonic Fibroblasts (MEFS). Our analysis revealed that these cells exist in two distinct states of migration characterized by differences in cell speed and persistence. Further analysis revealed that the Rho-family GTPase RhoG plays a role in establishing these two states. An important feature of our computational approach is that it provides a method for predicting the current migration state of an individual cell from time series data. Using this feature, we demonstrate that HeLa cells also exhibit two states of migration, and that these states correlate with differences in the spatial distribution of active Rac1.

cell biology↗

bioRxiv2017

Principles that govern competition or co-existence in Rho-GTPase driven polarization

Rho-GTPases are master regulators of polarity establishment and cell morphology. Positive feedback enables concentration of Rho-GTPases into clusters at the cell cortex, from where they regulate the cytoskeleton. Different cell types reproducibly generate either one (e.g. the front of a migrating cell) or several clusters (e.g. the multiple dendrites of a neuron), but the mechanistic basis for uni-polar or multi-polar outcomes is unclear. The design principles of Rho-GTPase circuits are captured by reaction-diffusion models based on conserved aspects of Rho-GTPase biochemistry. Some such models display rapid winner-takes-all competition between clusters, yielding a unipolar outcome. Other models allow prolonged co-existence of clusters. We derive a \"saturation rule\" general to all relevant models that governs the timescale of competition, and thereby predicts whether the system will generate uni-polar or multi-polar outcomes. We suggest that the saturation rule is a fundamental property of the Rho-GTPase polarity machinery, regardless of the specific feedback mechanism.

cell biology↗