Biology subjects

Choubey, S.

Publications and source records attributed to Choubey, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Surface-induced tau condensation generates a selective microenvironment around microtubules

Tau is a neuron-specific microtubule-associated protein that can self-associate into pathological insoluble aggregates or phase separate into condensates whose (patho)physiological role is debated. Recent studies suggest that intracellular surfaces can locally promote biomolecular condensation, even at low molecular concentrations. While microtubules in neurons provide an abundant tau-interaction surface, their role in tau phase separation remains unclear. Through a dialogue between experiments and theory, we demonstrate that tau forms multilayered condensates on microtubules at physiological concentrations via a prewetting-like transition. Concomitant tau-microtubule and tau-tau interactions explain the experimentally observed cooperative binding of the innermost tau layer directly adsorbed to the microtubule. The formation of this layer is dictated by the spacing of tubulin dimers within the microtubule lattice. Additional tau layers, driven by tau-tau interactions and independent of lattice spacing, are finite in thickness and unstable away from the microtubule surface. While the microtubule-adsorbed tau can selectively restrict proteins from the microtubule surface, the multilayered tau condensates can recruit tau interactors, such as RNA or soluble tubulin, highlighting the distinct roles of the condensate layers. Our results suggest that a prewetting-like transition constitutes a general physical mechanism for organizing liquid-like biomolecular layers of defined composition on charged intracellular surfaces.

biophysics↗

bioRxiv2026

A Sensory Pathway Controls Motile Microcolony Formation and Spatial Organization in Gliding Bacteria

How cells transition between distinct modes of collective organization is a fundamental question in biology. We uncover the regulatory landscape governing the choice between bacterial swarming and zorbing. In Flavobacterium johnsoniae, zorbs are spherical, biofilm-like microcolonies that migrate collectively, whereas swarms are multilayered and ordered populations that expand planarly. We identify RgzAB, an unorthodox sensor kinase-response regulator pair governing this transition; suppressor and knockout mutants establish the importance of RgzA phosphorelay and RgzB as a downstream component. Transcriptomic and perturbation analyses link this switch to iron availability, connecting environmental sensing to collective behavior. Remarkably, RgzA activity also governs spatial organization within mixed populations: activated cells encapsulate wildtype neighbors into segregated co-zorbs, within which wildtype cells assemble into networks undergoing an abundance-dependent, percolation-like transition. Together, these findings reveal that one sensory circuit governs bacterial collectives at nested scales, from motility choice to the internal architecture of the collective.

microbiology↗

bioRxiv2026

Leiomodin 2 functions as a processive pointed-end elongator of actin filaments

The actin cytoskeleton drives essential processes like cell migration and muscle contraction. While barbed-end polymerization is well-established, pointed-end elongation was long considered impossible in vivo. Here, we demonstrate that Leiomodin 2 (Lmod2), which localizes to thin-filament pointed ends (PEs) in striated muscle cells, functions as the first identified eukaryotic processive actin polymerase. Single-molecule and single-filament imaging reveal that Lmod2 stably associates with PEs in vitro, enabling elongation even in the presence of high profilin concentrations found in the cytoplasm that otherwise would cause depolymerization of free PEs. We find that both processivity and elongation rate of Lmod are dependent on its WH2 domain. Remarkably, human dilated cardiomyopathy-associated mutations in Lmod2 greatly reduce Lmod2s PE elongation activity, providing a potential mechanism for disease progression, underscoring the essential role of its actin polymerase activity in formation and maintenance of muscle sarcomeres.

biochemistry↗

bioRxiv2026

A moments-based approach for inferring mechanisms of transcriptional regulation using nascent RNA data

AbstractUnraveling the kinetics of transcriptional regulation is a central challenge in molecular biology. Traditional inference methods based on cell-to-cell mRNA variability are limited because mRNA levels reflect not only transcriptional activity but also post-transcriptional processes. Recent advances in quantifying nascent RNAs across isogenic cells provide a more direct readout of transcriptional dynamics. Here, we present a moments-based theoretical framework that leverages nascent RNA data to infer transcriptional kinetics. We derive closed-form expressions for the first two time-dependent moments of nascent RNA across three canonical transcription models and demonstrate that nascent RNA exhibits distinct transient overshoots and higher Fano factors compared to mature mRNA. Through analysis of synthetic datasets, we show that these features enable more accurate estimation of transcriptional kinetics and allow reliable model selection, distinguishing single from multi-pathway promoter activation with over 75% accuracy even from small sample sizes. We further validated our model selection method using real experimental transcription datasets in yeast and E. coli cells, with nascent RNA demonstrating superior performance in identifying cross-talk pathway regulation. Our framework thus offers a computationally efficient and mechanistically informative approach for decoding transcriptional dynamics, emphasizing the advantages of time-dependent nascent RNA measurements over mature mRNA in capturing the true kinetics of transcriptional regulation.

biophysics↗

bioRxiv2025

Phase separation as a tunable regulator of canonical gene regulatory motifs

Gene regulatory networks govern essential cellular processes such as signal transduction, metabolism, and cell fate control. Within these networks, canonical motifs such as genetic toggle switches and repressilators serve as building blocks that generate bistable and oscillatory behaviors. An open question is how cells regulate the dynamical behavior of such motifs, both in natural contexts and in synthetic systems. Recent studies highlight phase separation of transcription factors (TFs) and nucleic acids as a key organizing principle of intracellular biochemistry. In this work, we explore how phase separation of TFs influences the dynamics of toggle switch and repressilator using mean-field theory and stochastic simulations. Our mean-field analysis reveals that phase separation reshapes motif dynamics by altering fixed-point stability and basin geometry in toggle switch, and by altering oscillatory cycles in repressilator. A key finding for both motifs is that when multiple TFs undergo phase separation, the one with the lowest saturation concentration for phase separation dominates system dynamics. Interestingly, stochastic simulations show that the impact of phase separation on fluctuations (or noise) in the abundance of transcription factors within network motifs is architecture-dependent and sharply contrasts with its buffering effect on noise in the expression of isolated genes. Overall, our results show that biomolecular phase separation acts as a physical principle for tuning stability and noise in gene regulatory networks, providing new insights into cellular decision-making.

biophysics↗

bioRxiv2025

Membrane Interfacial Potential Governs Surface Condensation andFibrillation of α-Synuclein in Neurons

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are essential for cellular organization. -Synuclein, an amyloidogenic protein linked to Parkinsons Disease (PD), undergoes phase separation at high concentrations, but the influence of lipid membranes on this process remains unclear. Here, combining in vitro reconstitution, cell biology, and simulations, we show that membranous interfaces promote -Synuclein condensation at physiologically relevant sub-critical concentrations ([~]10 nM) without crowding agents. Notably, condensation occurs only on membranes with a specific stoichiometry of lipids, underscoring the role of interfacial potential. These condensates serve as nucleation sites for fibril formation, leading to membrane deformation and rupture. A lattice gas model reveals this behavior as a prewetting-like transition, where an attractive membrane induces local phase separation below the bulk saturation concentration. Indeed altering interfacial potential by lipid composition and membrane depolarization not only drastically changes -Synuclein puncta size and number but also triggers their release from neurons. These findings reveal the crucial role of lipid membrane interfaces in regulating -Synuclein condensation, aggregation and release, shedding light on a potential mechanism of their cell-to-cell propagation during neurodegeneration.

biophysics↗

bioRxiv2025

Sequence-dependent co-condensation of Lsr2 with DNA elucidates the mechanism of genome compaction in Mycobacterium tuberculosis

The xenogeneic silencer protein Lsr2 from Mycobacterium tuberculosis plays a critical role in its survival and pathogenesis. Lsr2 is a nucleoid-associated protein that interacts with DNA in vivo and regulates many genes. Purified Lsr2 forms nucleoprotein filaments with DNA molecules leading to highly compacted DNA conformations. However, the physical mechanism underlying Lsr2-mediated DNA compaction, resulting in gene regulation, remains elusive. We employed a combination of biochemical assay, single-molecule imaging, and molecular dynamics simulations to investigate the governing principles of Lsr2-mediated DNA compaction. We show that, while Lsr2 alone undergoes phase separation, addition of DNA substantially lowers the required concentration for its phase separation. Strikingly, our single-molecule and simulation data establish that Lsr2 forms condensates with long stretches of AT-rich DNA, providing strong evidence for sequence-dependent co-condensation. This observation is contrary to the classical view of sequence-dependent binding of individual protein molecules to DNA, our findings rather suggest that protein-DNA co-condensates sense the average binding energy landscape. We present a physical model for Lsr2-mediated DNA compaction and gene regulation, describing a novel mechanism for NAP-mediated genome organization in bacteria.

biophysics↗

bioRxiv2024

A generalized theoretical framework to investigate multicomponent actin dynamics

The length of actin filaments is regulated by the combined action of hundreds of actin-binding proteins. While the roles of individual proteins are well understood, how they combine to regulate actin dynamics in vivo remains unclear. Recent advances in microscopy have enabled precise, high-throughput measurements of filament lengths over time. However, the absence of a unified theoretical framework has hindered a mechanistic understanding of the multicomponent regulation of actin dynamics. To address this, we propose a general kinetic model that captures the combined effects of multiple regulatory proteins on actin dynamics. We provide closed-form expressions for both time-dependent and steady-state moments of the filament length distribution. Our framework not only differentiates between various regulatory mechanisms but also serves as a powerful tool for interpreting current data and driving future experiments.

biophysics↗

bioRxiv2024

Direct contact of the bacterial surface induces phase separation in the host phagosome membrane

Mycobacterium tuberculosis (Mtb) establishes intracellular niches by remodeling host membranes into either spacious or compact phagosomes, yet how direct bacterial contact within these distinct compartments facilitates bacterial egress remains unknown. Using fixed cell imaging, in vitro reconstitution of phagosome-like vesicles, and numerical simulations, we uncover that mycobacterial load and its direct contact determines phagosome fate by driving membrane bending, lipid wrapping and phase separation. Low-to-moderate load induces membrane vesiculation, generating compact phagosome-like structures. High bacterial load drives a scaffold-like architecture that primes compartments for rupture via complete lipid phase separation and changes in the membranes bending and area stretch moduli, rendering it more deformable. Notably, physical contact synergizes with the virulence factor ESAT-6, amplifying its membrane-deforming activity to disrupt phagosomal integrity. We propose that mycobacteria actively switch spacious to compact phagosomal states by modulating host membrane mechanics--a process governed by the membrane-to-contact area ratio and proximity to lipid demixing point. These findings reveal a biophysical switch in pathogen-driven membrane remodeling, with broad implications for understanding how intracellular pathogens manipulate host membranes to survive.

biophysics↗

bioRxiv2024

Real-time visualization reveals Mycobacterium tuberculosis ESAT-6 disrupts phagosome via fibril-mediated vesiculation

Mycobacterium tuberculosis (Mtb) evades host defense by hijacking and rupturing the phagosome, enabling it to escape to the host cytosol for its survival. ESAT-6, a secreted virulence protein of Mtb, is known to be critical for phagosome rupture. However, the mechanism of ESAT-6-mediated disruption of the phagosomal membrane remains unknown. Using in vitro reconstitution and numerical simulations, we discover that ESAT-6 polymerization remodels and vesiculates phagosomal membrane. In contrast to the pore formation triggered by a bilayer-spanning conformation, we find that the binding of ESAT-6 to the phagosomal membrane is shallow. Such shallow insertion leads to membrane shape transition leading to tubular and bud-like deformations on the membrane in a concentration-dependent manner, facilitated by the reduction in membrane tension and compressibility modulus. Strikingly, our observations suggest that ESAT-6 polymerizes in bulk and on the membrane, both in vitro and in macrophage. Numerical simulations demonstrate that growing fibrils generate both radial and tangential forces causing local remodeling and shape transition of the membrane. Using micropipette aspiration, we quantitatively show that ESAT-6 bound tensed membrane undergoes local changes in membrane curvature and lipid phase separation, also facilitated by the direct contact of the bacteria inside the phagosome. Nonetheless, the vesiculation of the buds is primarily driven by the forces exerted by the polymerization of ESAT-6. Such ESAT-6 mediated vesiculation induces apoptosis and host cell death in a concentration and time-dependent manner that promotes infection. Overall, the findings provide mechanistic insights into the long-standing question of phagosome disruption by Mtb for its escape.

biophysics↗

bioRxiv2024

Role of protein-protein interactions on model chromatin organization

The three-dimensional organization of chromatin is influenced by DNA-binding proteins, through specific and non-specific interactions. However, the role of DNA sequence and interaction between binding proteins in influencing chromatin structure is not yet fully understood. By employing a simple polymer-based model of chromatin, that explicitly considers sequence-dependent binding of proteins to DNA and protein-protein interactions, we elucidate a mechanism for chromatin organization. We find that: (1) Tuning of protein-protein interaction and protein concentration is sufficient to either promote or inhibit the compartmentalization of chromatin. (2) The presence of chromatin acts as a nucleating site for the condensation of the proteins at a density lower than in isolated protein systems. (3) The exponents describing the spatial distance between the different parts of the chromatin, and their contact probabilities are strongly influenced by both sequence and the protein-protein attraction. Our findings have the potential application of re-interpreting data obtained from various chromosome conformation capture technologies, thereby laying the groundwork for advancing our understanding of chromatin organization.

biophysics↗

bioRxiv2024

Decoding the role of DNA sequence on protein-DNA co-condensation

The eukaryotic genome is organized within the cell nucleus through three-dimensional compaction. The physical principles that govern genome organization in vivo remain less understood. Phase separation of protein and DNA has emerged as an attractive mechanism for reshaping chromatin and compacting the genome. In vitro studies have shed light on the biophysical principles of protein-DNA condensates driven by protein-protein and protein-DNA interactions. However, the role of DNA sequence and its impact on protein-DNA condensation remains elusive. Guided by experiments, this paper presents a simple polymer-based model of protein-mediated DNA condensation that explicitly incorporates the influence of DNA sequence on protein binding. Using coarse-grained Brownian dynamics simulations, we demonstrate that, in the case of a homogeneous DNA, only one condensate forms in equilibrium. In sharp contrast, DNA sequence heterogeneity can result in the coexistence of multiple condensates, giving rise to the formation of structures resembling pearl-necklaces. Interestingly, we observe that protein binding affinity of interfacial DNA governs the capillary forces arising from the protein-DNA condensates. To demonstrate the usefulness of our modeling framework, we compare the simulation results against published data for co-condensation of Dps, Sox2, and HP1. We find that while Dps exhibits sequence-independent binding, DNA sequence heterogeneity dictates the co-condensation of Sox2 and HP1 with DNA. Overall, the framework developed here can be harnessed to gain mechanistic insights into the role of DNA sequence on protein-DNA co-condensation and pave the way for developing a deeper understanding of genome organisation.

biophysics↗

bioRxiv2023

Governing principles of transcriptional logic out of equilibrium

Cells face a myriad of environments and signaling cues. In order to survive, adapt, and desvelop, cells respond to external and internal stimuli, by tightly regulating transcription. Transcriptional regulation involves combinatorial binding of a repertoire of transcription factors (TFs) to DNA, which often results in switch-like binary outputs, akin to logic gates. Recent experimental studies have demonstrated that transcription factor binding to DNA often involves energy expenditure, thereby driving the system out of equilibrium. The governing mechanistic principles of transcriptional logic out of equilibrium remain elusive. To this end, we employ a simple two-input model of transcription that explicitly considers the non-equilibrium binding of TFs to DNA. This simple model can accommodate both equilibrium and non-equilibrium mechanisms and allows for a comparative study of logic operations obtained in the two regimes. We find that, out of equilibrium, the regulatory function of two transcription factors gets altered in a mutually exclusive manner. Such behavior allows non-equilibrium regimes to recreate all the logic operations seen in equilibrium and create new logic operations inaccessible in equilibrium. Our findings demonstrate that cells attain a wider range of decision-making abilities by expending energy.

biophysics↗

bioRxiv2022

Temporal Regulation of Organelle Biogenesis

Organelle abundance is tightly regulated in eukaryotic cells in response to external stimuli. The underlying mechanisms responsible for this regulation remains less understood. Time-lapse imaging of fluorescently labelled organelles allow for counting individual organelle copies at a single-cell level. These experiments contain information about the time evolution of distribution of organelle number across a population of cells. To tap onto such data, we build upon a recently proposed kinetic model of organelle biogenesis that explicitly incorporates de novo synthesis, fission, fusion and degradation of organelles. Different limits of this general model correspond to distinct mechanisms of organelle biogenesis. We compute the first two moments of organelle number distribution for these different mechanisms. Interestingly, different mechanisms of biogenesis lead to qualitatively distinct temporal behavior of cell-to-cell variability (noise), thereby allowing us to discern between these mechanisms. Notably, noise in temporal organelle abundance exhibits strikingly more complex behaviour as compared to the steady state. Our modeling framework paves the way for extracting quantitative information about the dynamics of organelle biogenesis from time-lapse experiments that can measure organelle abundance at single-molecule resolution.

systems biology↗

bioRxiv2020

Tunability enhancement of gene regulatory motifs through competition for regulatory protein resources

Gene regulatory networks (GRN) orchestrate the spatio-temporal levels of gene expression, thereby regulating various cellular functions ranging from embryonic development to tissue home-ostasis. Some patterns called "motifs" recurrently appear in the GRNs. Owing to the prevalence of these motifs they have been subjected to much investigation both in the context of understanding cellular decision making and engineering synthetic circuits. Mounting experimental evidence suggest that 1) the copy number of genes associated with these motifs vary, and 2) proteins produced from these genes bind to decoy binding sites on the genome as well as promoters driving the expression of other genes. Together, these two processes engender competition for protein resources within a cell. To unravel how competition for protein resources affect the dynamical properties of regulatory motifs, we propose a simple kinetic model that explicitly incorporates copy number variation (CNV) of genes and decoy binding of proteins. Using quasi steady-state approximations, we theoretically investigate the transient and steady-state properties of three of the commonly found motifs: autoregulation, toggle switch and repressilator. While protein resource competition alters the timescales to reach the steady-state for all these motifs, the dynamical properties of toggle switch and repressilator are affected in multiple ways. For toggle switch, the basins of attraction of the known attractors are dramatically altered if one set of proteins bind to decoys more frequently than the other, an effect which gets suppressed as copy number of toggle switch is enhanced. For repressilators, protein sharing leads to emergence of oscillation in regions of parameter space that were previously non-oscillatory. Intriguingly, both the amplitude and frequency of oscillation are altered in a non-linear manner through the interplay of CNV and decoy binding. Overall, competition for protein resources within a cell provides an additional layer of regulation of gene regulatory motifs.

systems biology↗

bioRxiv2020

Efficacy of information transmission in cellular communication

Inter and intra-cellular signaling are essential for individual cells to execute various physiological tasks and accurately respond to changes in their environment. Signaling is carried out via diffusible molecules, the transport of which is often aided by active processes that provide directional advection. How diffusion and advection together impact the accuracy of information transmission during cell signaling remains less studied. To this end, we study a one-dimensional model of cell signaling and compute the mutual information (MI) as a measure of information transmission. We find that the efficacy of the information transmission improves with advection only when the system parameters result in Peclet number greater than one. Intriguingly, MI exhibits nontrivial scaling with the Peclet number, characterized by three distinct regimes. We demonstrate that the observed dependence of MI on the transport properties of signaling molecules has important consequences on cellular communication.

biophysics↗

bioRxiv2020

Surface condensation of a pioneer transcription factor on DNA

In the last decade, extensive studies on the properties of non-membrane-bound compartments in the cellular cytoplasm have shown that concepts in phase separation drawn from physical chemistry can describe their formation and behaviour1-4. Current evidence also suggests that phase separation plays a role in the organization inside the cell nucleus5-8. However, the influence and role of DNA on the physical chemistry of phase separation is not well understood. Here, we are interested in the role of interactions between phase separating proteins and the DNA surface. The interaction of liquid phases with surfaces has been extensively studied in soft matter physics, in the context of macroscopic surfaces and non-biological liquids9-11. The conditions in the nucleus are different from those studied in conventional soft matter physics because DNA with a diameter of about 2 nm12 provides a microscopic surface, and liquid-like phases are complex mixtures of proteins subject to a myriad of biochemical modifications13. Transcriptional condensates, which are thought to serve as regulatory hubs in gene expression14-21, provide an accessible system to investigate the physics of condensates that emerge from DNA-protein and protein-protein interactions. These condensates are typically small22, and the mechanisms that determine their size are unknown. Whether they can be understood as phase separated compartments has been subject to debate23-26. Here, we use optical tweezers to directly observe the condensation of the pioneer transcription factor Klf427,28 on DNA in vitro. We demonstrate that Klf4 forms microphases that are enabled by interaction with the DNA surface. This sets their typical size and allows them to form below the saturation concentration for liquid-liquid phase separation. We combine experiment with theory to show that these microphases can be understood as forming by surface condensation on DNA via a switch-like transition similar to prewetting. Polymer surface mediated condensation reconciles several observations that were previously thought to be at odds with the idea of phase separation as an organizing principle in the nucleus.

biophysics↗

bioRxiv2019

Probing mechanisms of transcription elongation through cell-to-cell variability of RNA polymerase

The process of transcription initiation and elongation are primary points of control in the regulation of gene expression. While biochemical studies have uncovered the mechanisms involved in controlling transcription at each step, how these mechanisms manifest in vivo at the level of individual genes is still unclear. Recent experimental advances have enabled single-cell measurements of RNAP molecules engaged in the process of transcribing a gene of interest. In this manuscript, we use Gillespie simulations to show that measurements of cell-to-cell variability of RNAP numbers and inter-polymerase distances can reveal the prevailing mode of regulation of a given gene. Mechanisms of regulation at each step, from initiation to elongation dynamics, produce qualitatively distinct signatures which can further be used to discern between them. Intriguingly, depending on the initiation kinetics, stochastic elongation can either enhance or suppress cell-to-cell variability at the RNAP level. To demonstrate the value of this framework, we analyze RNAP number distribution data for ribosomal genes in S. cerevisiae from three previously published studies and show that this approach provides crucial mechanistic insights into the transcriptional regulation of these genes.\n\nAuthor SummaryThe process of transcription comprises many distinct steps and understanding the regulation of each of these steps provides insight into how the levels of gene expression are controlled in the cell. In this manuscript, we use stochastic simulations to explore how regulation at the level of elongation and initiation together influences the distribution of actively transcribing RNAP on a gene. We find that each of these steps of regulation leaves a distinct imprint on the gene-to-gene variability of number of actively transcribing RNAP on the gene and their inter-RNAP distances. Using these results, we analyze recent experimental data of transcribing RNAP distributions and find that the perturbations in these studies primarily impact the transcription initiation dynamics of the genes.

cell biology↗