Biology subjects

Golding, I.

Publications and source records attributed to Golding, I..

12 recordsLinked to original sources

bioRxiv2026

Nuclear speckles comprise hierarchical nanoscale interaction networks that shape molecular dynamics

Biomolecular condensates organize essential cellular processes, but how their internal architecture regulates molecular behavior in living cells remains poorly understood. Here, we use MINFLUX nanoscopy for quantitative single-molecule imaging and live-cell tracking to resolve the organization and dynamics of specific macromolecules within nuclear speckles. Rather than behaving as uniform liquid-like compartments, nuclear speckles are hierarchically organized into dynamic nanoscale interaction networks. Proteins and RNAs assemble into molecular clusters that generate transient interaction nanodomains, where individual molecules alternate between diffusion and confinement while exploring higher-order networks. Physiological and genetic perturbations selectively remodel molecular clustering and nanodomain organization, and these changes are not consistently coupled to changes in the abundance of the measured components. Together, these findings reveal a multiscale organization of nuclear speckles in which molecular clustering, transient confinement, and dynamic exchange represent distinct but interconnected features of condensate architecture in living cells.

cell biology↗

bioRxiv2026

Temporal dynamics of RNA phage infection reveal bacterial recovery despite ongoing viral replication

Bacterial defenses against DNA phages are well documented, yet responses to RNA phages remain largely unexplored. Here, we characterize the population dynamics and host responses of Escherichia coli infected by the lytic RNA phage MS2 across multiple timescales, using bulk and single-cell transcriptomics, fluorescence microscopy, whole-genome sequencing, and genetic perturbations. Despite rapid viral takeover and extensive cell death, the bacterial population subsequently rebounds, revealing an unusual state in which host proliferation persists under intense phage pressure. Early in infection, heterogeneous subpopulations are evident, including one with low or undetectable viral transcript levels, low expression of the F-plasmid genes encoding the F-pilus (the MS2 receptor), and a distinct metabolic state. Strikingly, the rebounding population carries MS2 RNA in the great majority of cells, with evidence for active viral replication and continued cell division despite high viral loads. This rebound coincides with strong induction of biofilm-associated genes, including the pga operon, which synthesizes the biofilm-matrix exopolysaccharide PNAG. Deletion of pgaA causes earlier population decline and reduced population survival, indicating that pga-dependent matrix production delays viral lysis. Over longer timescales, phage-exposed populations show heterogeneous F-plasmid prevalence and plasmid-wide expression, consistent with dynamic cycling between plasmid-bearing and plasmid-free states: MS2 selects against F-plasmid-bearing cells, whereas conjugation may restore plasmid carriage. We propose that the biofilm-associated matrix delays viral lysis relative to continued cell division, long enough for rare F-plasmid-free daughter cells to arise and expand. Our results suggest that bacterial survival is determined by a dynamic race between viral lysis and host-cell division.

microbiology↗

bioRxiv2026

Cell Growth and Division Shape mRNA-Protein Correlations

1Correlations between cellular variables, such as gene-expression levels, provide insights into regulatory mechanisms. We focus here on correlations between mRNA and protein levels and re-examine previously derived analytical predictions. We test this prediction on single-cell E. coli data and see substantial disagreement. We hypothesize that this discrepancy arises from the assumption of constant cell volume and develop a theoretical framework for mRNA-protein correlations in growing and dividing cells. Within this framework, we derive an analytical expression for mRNA- protein correlations and show that explicit incorporation of growth and division substantially alters these correlations. The resulting relation is invariant to upstream transcriptional dynamics, and we validate it using stochastic simulations across multiple gene-regulatory architectures. Finally, we show that the derived predictions are consistent with the E. coli data.

biophysics↗

bioRxiv2026

Single-phage profiling illuminates viral individuality during cell fate determination

The choice between cell death (lysis) and viral dormancy (lysogeny) following bacteriophage infection serves as a founding paradigm for the emergence of cellular heterogeneity in a genetically uniform population. The determination of host fate arises through the stochastic transcription from multiple viral genomes present within each cell, but this activity remains hidden from empirical interrogation, which typically stops at the whole-cell level. Here we use parallel sequential fluorescence in situ hybridization (par-seqFISH), followed by spatial clustering of phage-encoded transcripts within each cell, to profile the transcriptional activity of individual phages during synchronized infection of Escherichia coli (E. coli) by bacteriophage lambda. At the whole-cell level, transcription kinetics capture the developmental choice between lysis and lysogeny, and further demonstrate that viral replication is required for the emergence of diverging fate decisions. Zooming in to the single-phage level illuminates an individuality of viral activity during infection. We find that, while cells pursuing lysogeny display consensus activity of all in-habiting phages, lytic cells may contain phages that exhibit lysogenic activity. These findings support an earlier suggestion that consensus among coinfecting phages is required for cell dormancy. More broadly, our results highlight the need to identify how whole-cell behavior emerges from the activity of physically distinct copies of the same genetic circuit.

systems biology↗

bioRxiv2025

Cytoplasmic localization of the mRNA encoding actin regulator, Serendipity-α, promotes adherens junction assembly and nuclear repositioning

The subcellular localization of mRNAs is conserved from prokaryotes to humans. In Drosophila embryos [~]70% of mRNAs localize to specific sites in the cytoplasm, but the functional significance of this mRNA localization is largely unknown. During the process of embryo cellularization, mRNA encoding Serendipity- (Sry-), an actin filament (F-actin) binding protein, moves apically, concentrating near centrosomes. Transport is mediated by the Egl/BicD/Dynein complex and requires two stem loops in the 3UTR of the mRNA, which serve as localization signals. mRNA localization is dispensable for Sry- function at cleavage furrows in early cellularization but is necessary for repositioning nuclei in late cellularization. Sry- protein promotes assembly of cortical F-actin and apical spot adherens junctions (AJs) in late cellularization, and these AJs contribute to nuclear repositioning. We suggest that mRNA localization restricts cytoskeletal functions in late cellularization to regulate nuclear repositioning in preparation for the tissue morphogenesis events that immediately follow.

cell biology↗

bioRxiv2025

Reducing Cofilin dosage makes embryos resilient to heat stress

In addition to regulating actin dynamics, Cofilin also senses and responds to physiological stress and can determine cell survival outcomes. Yet, the full picture of Cofilin's role in stress response is lacking. Here, we used imaging and RNA-seq methods to show that exposing early Drosophila melanogaster embryos to either acute or chronic heat stress (32{degrees}C) induces a Cofilin-mediated Actin Stress Response (ASR) and leads to upregulation of genes associated with both heat shock and Endoplasmic Reticulum (ER) unfolded protein responses. Reducing cofilin gene dosage (cofilin+/-) in heat-stressed embryos modulates all observed stress responses and partially rescues embryo survival. Unexpectedly we find that heat shock- and ER- stress response modulation arises because non-stressed cofilin+/- embryos already show upregulation of heat shock- and ER- stress response genes, prior to heat exposure. Our data support a model whereby cofilin heterozygosity activates specific stress responses associated with reduced protein homeostasis, thus priming embryos to be more resilient when they encounter subsequent heat stress. We conclude that Cofilin dosage serves as a determinant of stress outcomes, impacting both the actin cytoskeleton and inducible stress response pathways. In the embryo, we identify Cofilin as a novel link between inducible stress response and thermotolerance.

developmental biology↗

bioRxiv2023

Using population dynamics to count bacteriophages and their lysogens

Traditional assays for counting bacteriophages and their lysogens are labor-intensive and highly perturbative to the host cells. Here, we present a high-throughput infection method where all steps--cell growth, viral encounters, and post-infection recovery--take place in a microplate reader, and the growth dynamics of the infected culture are measured continuously using the optical density (OD). We find that the post-infection dynamics are reproducible and interpretable. In particular, the OD at which the culture lyses scales linearly with the logarithm of the initial phage concentration, providing a way of measuring phage numbers in unknown samples over nine decades and down to single-phage sensitivity. Interpreting the measured dynamics using a mathematical model for the coupled kinetics of phages and bacteria further allows us to infer the rates of viral encounters and cell lysis. Adding a single step of antibiotic selection provides the ability to measure the rate of host lysogenization. To demonstrate the application of our assay, we characterized the effect of bacterial growth rate on the propensity of lambda phage to lysogenize E. coli. When infected by a single phage, the probability of lysogenization is found to decrease approximately exponentially with the host growth rate. In growing, but not in stationary, cells, the propensity to lysogenize increases ~50-fold when multiple phages co-infect the cell. These findings illuminate how host physiology feeds into the lysis/lysogeny decision circuit, and demonstrate the utility of high-throughput infection to interrogating phage-host interactions.

microbiology↗

bioRxiv2023

Co-infecting phages impede each other's entry into the cell

Bacteriophage lambda tunes its propensity to lysogenize based on the number of viral genome copies inside the infected cell. Viral self-counting is believed to serve as a way of inferring the abundance of available hosts in the environment. This interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). However, here we show this premise to be untrue. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, followed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of individual phage entries decrease with MOI. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage landing, evidenced by compromised membrane integrity and loss of membrane potential. The dependence of phage entry dynamics on the surrounding medium is found to result in a strong impact of environmental conditions on the infection outcome, while the protracted entry of co-infecting phages increases the cell-to-cell variability in infection outcome at a given MOI. Our findings demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection.

microbiology↗

bioRxiv2023

Physiological stress drives the emergence of a Salmonella subpopulation through ribosomal RNA regulation

Bacteria undergo cycles of growth and starvation, to which they must adapt swiftly. One important strategy for adjusting growth rates relies on ribosomal levels. While high ribosomal levels are required for fast growth, their dynamics during starvation remain unclear. Here, we analyzed ribosomal RNA (rRNA) content of individual Salmonella cells using Fluorescence In-Situ Hybridization (rRNA-FISH). During the transition from exponential to stationary phase we measured a dramatic decrease in rRNA numbers only in a subpopulation, resulting in a bimodal distribution of cells with high and low rRNA content. We showed that the two subpopulations are phenotypically distinct when subjected to nutritional upshifts. Using a transposon screen coupled with rRNA-FISH, we identified two mutants acting on rRNA transcription shutdown and degradation, that abolished the formation of the subpopulation with low rRNA content. Our work suggests that Salmonella employs a bet-hedging strategy in regulating ribosomal levels that may be beneficial for survival.

microbiology↗

bioRxiv2021

Bacteriophage self-counting in the presence of viral replication

When host cells are in low abundance, temperate bacteriophages opt for dormant (lysogenic) infection. Phage lambda implements this strategy by increasing the frequency of lysogeny at higher multiplicity of infection (MOI). However, it remains unclear how the phage reliably counts infecting viral genomes even as their intracellular number increases due to replication. By combining theoretical modeling with single-cell measurements of viral copy number and gene expression, we find that, instead of hindering lambdas decision, replication facilitates it. In a nonreplicating mutant, viral gene expression simply scales with MOI rather than diverging into lytic (virulent) and lysogenic trajectories. A similar pattern is followed during early infection by wildtype phage. However, later in the infection, the modulation of viral replication by the decision genes amplifies the initially modest gene expression differences into divergent trajectories. Replication thus ensures the optimal decision--lysis upon single-phage infection, lysogeny at higher MOI.

systems biology↗

bioRxiv2020

Flagellar dynamics reveal large fluctuations and kinetic limit in the Escherichia coli chemotaxis network

The Escherichia coli chemotaxis network, by which bacteria modulate their random run/tumble swimming pattern to navigate their environment, must cope with unavoidable number fluctuations ("noise") in its molecular constituents like other signaling networks. The probability of clockwise (CW) flagellar rotation, or CW bias, is a measure of the chemotaxis networks output, and its temporal fluctuations provide a proxy for network noise. Here we quantify fluctuations in the chemotaxis signaling network from the switching statistics of flagella, observed using time-resolved fluorescence microscopy of individual optically trapped E. coli cells. This approach allows noise to be quantified across the dynamic range of the network. Large CW bias fluctuations are revealed at steady state, which may play a critical role in driving flagellar switching and cell tumbling. When the network is stimulated chemically to higher activity, fluctuations dramatically decrease. A stochastic theoretical model, inspired by work on gene expression noise, points to CheY activation occurring in bursts, driving CW bias fluctuations. This model also shows that an intrinsic kinetic ceiling on network activity places an upper limit on activated CheY and CW bias, which when encountered suppresses network fluctuations. This limit may also prevent cells from tumbling unproductively in steep gradients.

biophysics↗

bioRxiv2019

Stochastic simulation platform for visualization and estimation of transcriptional kinetics

We present an implementation of the Gillespie algorithm that simulates the stochastic kinetics of nascent and mature RNA. Our model includes two-state gene regulation, RNA synthesis initiation and stepwise elongation, release to the cytoplasm, and stepwise degradation, a granular description currently tractable only by simulation. To facilitate comparison with experimental data, the algorithm predicts fluorescent probe signals measurable by single-cell RNA imaging. We approach the inverse problem of estimating underlying parameters in a five-dimensional parameter space and suggest optimization heuristics that successfully recover known reaction rates from simulated gene expression turn-on data. The simulation framework includes a graphical user interface, available as a MATLAB app at https://data.caltech.edu/records/1287.

biophysics↗