Biology subjects

Dunlap, D.

Publications and source records attributed to Dunlap, D..

14 recordsLinked to original sources

bioRxiv2026

Biophysical Characterization of ParBS Condensates suggests a physical mechanism for segregation

The ParABS system orchestrates chromosome segregation in many bacterial species. The centromere-like parS sites serve as nucleation points for the binding of the ParB protein. Subsequent diffusion on adjacent, non-specific DNA regions (spreading) in the presence of CTP and binding of more ParB molecules along with DNA looping via ParB-ParB interactions bring distal parts of the chromosome into proximity. ParB interaction with the ParA-ATPase motor protein, then, drives genomic segregation. It has been shown that in some bacterial species, the ParB-parS complex undergoes phase separation into a condensate. However, the physico-chemical properties of such condensates and their response to forces, such as those they may face in the cell, have not yet been characterized. Performing turbidity measurements in the presence of CTP, various concentrations of DNA and physiological mono and divalent salt, it was shown that Mg2+ facilitates, while K+ concentrations higher than ~20 mM disfavor, condensate formation. Micro rheology measurements showed that condensates of ParB and DNA including parS sites (ParB-parS DNA) in the presence of CTP, are viscoelastic with a viscosity at Troom of ~5 Pa s and able to respond to deformations with a network relaxation time of 0.1 s. Additionally, fluorescence combined with force spectroscopy showed that mechanical disruption of ParB-DNA condensates in the presence of CTP requires 5-7.5 pN of tension in the DNA, which is lower than the force required to stall a molecular motor such as RNA polymerase, but higher than the force required for the relocation of chromosomes and plasmids during segregation. These results support the idea that ParB-parS condensates dynamically rearrange at the molecular level while maintaining the cohesion necessary to sustain the drag force of segregation without interfering with genomic transactions. This physical mechanism could be the basis for the critical role of ParB-parS condensates in organizing and partitioning bacterial chromosomes.

biophysics↗

bioRxiv2026

Practical quantification of immunohistochemistry antigen concentrations and reaction-diffusion parameters

The immunohistochemistry (IHC) methods widely used in diagnostic medicine and biomedical research are kinetically complex reaction-diffusion processes that, ideally, produce stain intensities correlated with the local antigen concentration. Yet after 75 years of use, practical theoretical tools to rigorously plan and interpret IHC experiments are still lacking. Because modeling the reactions requires time-consuming computer simulation, impractical for regular use, most protocols are optimized empirically, without detailed knowledge of the reaction rates and antigen-antibody equilibria. The resulting stain intensities can be calibrated against standards with known antigen abundance, but they are typically not interpretable in terms of chemical antigen concentrations. To address these limitations, we developed a fast interpolation method to model reaction-diffusion behavior, and experimental methods to characterize IHC kinetic parameters in formalin-fixed paraffin-embedded (FFPE) samples. Used together, these allow experimental measurement of both the chemical concentration of antigen in the sample and the reaction-diffusion parameters consistent with the assay results. Results show 1) direct immunofluorescent detection has low nanomolar sensitivity with >1000-fold dynamic range, and 2) antibody diffusion rates in FFPE samples can be >1000-fold slower than in aqueous solutions, producing diffusion-limited conditions in which the IHC reaction time course may depend on the sample antigen concentration. Awareness of these details is necessary to avoid potential underestimation of both the absolute and relative antigen concentrations in different samples that may occur if staining is stopped before reaching equilibrium. Software tools are provided to allow users to rapidly model IHC reaction time courses and to fit experimental time course data with candidate reaction parameters. The principles described here apply equally to other tissue-based "spatial omics" analyses and should be considered when designing and interpreting experiments requiring any macromolecule to diffuse into and react in a tissue section. SIGNIFICANCEThe theoretical and experimental framework described here advances IHC staining from a qualitative or semi-quantitative method towards a more rigorously quantitative assay. The practical ability to predict IHC reaction kinetics and fit reaction parameters to experimental data has the potential to advance IHC applications in diagnostic medicine and biomedical research in three ways: 1) interpretation of experimental and diagnostic samples stained under different conditions can be more objective, facilitating comparison of results from different protocols and different laboratories; 2) IHC staining can be interpreted as molar chemical antigen-antibody concentrations calculated from the reaction parameters measured in the studied sample; 3) the correlation between antigen concentration and biological behavior can be examined more reliably. Practical software tools are provided.

pathology↗

bioRxiv2026

Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon

Conjugative plasmids drive bacterial evolution and antibiotic resistance spread, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that despite sharing similar DNA motifs and a preference for negative supercoiling, Sfx and H-NS occupy distinct niches: Sfx binds weakly to the chromosome but is enriched on the R6K vir operon, from which H-NS is excluded. We hypothesize that this selective targeting is mediated by Sfx-vir interactions and phase separation. We show that Sfx binding to vir DNA critically depends on DNA topology but not on the target location. Our results suggest that Sfx phase separates with R6K to ensure its preferential recruitment to the plasmid DNA and forms stable nucleoprotein filaments that are impermeable to competitors. These findings reveal how histone-like proteins can partition the genome into distinct regulatory niches, a strategy likely mirrored across all life. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/707533v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1ee08a9org.highwire.dtl.DTLVardef@7ab423org.highwire.dtl.DTLVardef@1c89543org.highwire.dtl.DTLVardef@1bfa132_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Recurrent RNA-lipoplex vaccination is required to sustain functional tumor-infiltrating neoantigen-specific CD8 T cells and therapeutic efficacy

Cancer vaccines induce durable, polyepitopic T cell responses, and show promising clinical benefit in adjuvant settings, yet they are largely ineffective in advanced disease. Using a clinically relevant RNA-lipoplex vaccine, we investigated the efficacy constraints in a preclinical model. Vaccination remodeled the tumor microenvironment (TME), increasing T cell infiltration and promoting a proinflammatory myeloid compartment. This was associated with complete regression of smaller, immature tumors, but only delayed growth of larger, established tumors. While vaccine-induced T cells were long-lived and functional in peripheral tissues, intratumoral T cells declined rapidly in abundance, diversity, and function, reverting to a prevaccine-like state. scRNA-seq suggested that this was driven by a pro-apoptotic program, with surviving T cells showing signatures of cellular stress and impaired activation. Importantly, recurrent vaccination replenished functional T cells in the TME and enhanced efficacy. These findings highlight the importance of optimizing vaccine schedules and tailoring therapeutic strategies to tumor stage.

immunology↗

bioRxiv2023

Force and the α-C-terminal domains bias RNApolymerase recycling

After RNA polymerase (RNAP) reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA before restarting RNA synthesis from the previous or a different promoter. We monitored such secondary transcription using magnetic tweezers to determine the effect of low forces, protein roadblocks, and transcription factors. Up to 60% of RNAPs diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 ({sigma}70) likely remained bound to these sliding RNAPs, enabling recognition of secondary promoters and additional rounds of transcription, and the addition of elongation factor NusG, which competes with{sigma} 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, RNAP blocked by a DNA-bound lac repressor could slowly re-initiate transcription at the block-age and was not affected by NusG, suggesting a{sigma} -independent pathway. Force biased the frequency of encounters between convergent or divergent promoters such that transcription was repetitive only from the promoter opposing the direction of force. Sliding RNAP recognized and could subsequently re-initiate from promoters in either orientation. However, deletions of the -C-terminal domains severely limited the ability of RNAP to turn around.

biophysics↗

bioRxiv2023

Lung-innervating neurons expressing Tmc3 can induce broncho-constriction and dilation with direct consequences for the respiratory cycle

Sensory neurons of the vagal ganglia (VG) innervate lungs and play a critical role in maintaining airway homeostasis. However, the specific VG neurons that innervate lungs, and the mechanisms by which these neurons sense and respond to airway insults, are not well understood. Here, we identify a subpopulation of lung-innervating VG neurons defined by their expression of Tmc3. Single cell transcriptomics illuminated several subpopulations of Tmc3+ sensory neurons, revealing distinct Piezo2- and Trpv1-expressing subclusters. Furthermore, Tmc3 deficiency in VG neurons leads to global and subcluster specific transcriptional changes related to metabolic and ion channel function. Importantly, we show that broncho-constriction and dilation can be modulated through inhibition or activation of Tmc3+ VG neurons resulting in a decrease or increase of end-expiratory lung volume, respectively. Together, our data show that Tmc3 is a marker of lung-innervating neurons and may play a pivotal role in maintaining fundamental inspiratory and expiratory processes. SignificanceHarnessing the neuronal mechanisms that regulate lung function offers potential alternatives to existing corticosteroid treatment regimens for respiratory illness associated with acute bronchoconstriction including asthma, COPD, and emphysema. Our findings define Transmembrane channel-like 3, Tmc3, as a marker of lung-innervating sensory neurons, identify distinct subpopulations of Tmc3+ neurons with unique transcriptional profiles, and show that activation or inhibition of these neurons has a significant impact on airway function. Our work highlights potential avenues of novel targeted intervention in respiratory conditions driven by dysfunctional neuronal reflexes.

neuroscience↗

bioRxiv2023

The renal capsule: a vibrant and adaptive cell environment of the kidney in homeostasis, disease and aging

The kidney is a complex organ that governs many physiological parameters. It is roughly divided into three parts, the renal pelvis, medulla, and cortex. Covering the cortex is the renal capsule, a serosal tissue that provides protection and forms a barrier for the kidney. Serosal tissues of many organs have been recently shown to play a vital role in homeostasis and disease. Analyses of the cells that reside in these tissues have identified distinct cell types with unique phenotypes. Here, we characterized this niche and found that it is mainly comprised of fibroblasts and macrophages, but also includes other diverse cell types. Characterizing renal capsule-associated macrophages, we found that they consist of a distinct subset (i.e., TLF+ macrophages) that is nearly absent in the kidney parenchyma. Injury, disease, and other changes that involve the kidney, affected the cell composition of the renal capsule, indicating its dynamic response to changes within the organ parenchyma. Lastly, we studied age-related changes in the renal capsule and found that aging affected the cell composition and inflammatory phenotype of macrophages, increased CD8 T cells and other lymphocyte counts, and promoted a senescence-associated phenotype in fibroblasts. Taken together, our data illustrate the complexity and heterogeneity of the renal capsule and its underlying changes during aging and disease, improving our understanding of the kidney serosa that may be valuable for novel renal therapies.

immunology↗

bioRxiv2023

Transcription Through Roadblock Systems Reveals a Hybrid Transit Mechanism

RNA polymerases (RNAPs) must transit through protein roadblocks to produce full-length RNAs. Here we report real-time measurements of Escherichia coli (E. coli) RNAP passage through different barriers. As intuitively expected, assisting forces facilitated, and opposing forces hindered, RNAP passage through LacI bound to natural operator sites. Force-dependent differences were significant at magnitudes as low as 0.2 pN and were abolished in the presence of GreA, which rescues backtracked RNAP. In stark contrast, opposing forces promoted passage when the rate of backtracking was comparable to, or faster than the rate of dissociation of the roadblock, particularly in the presence of GreA. Our experiments and simulations indicate that RNAP may transit after roadblocks dissociate, or undergo cycles of backtracking, recovery, and ramming into roadblocks to pass through. We propose that such reciprocating motion also enables RNAP to break protein-DNA contacts holding RNAP back during promoter escape and RNA chain elongation, facilitating productive transcription in vivo.

biophysics↗

bioRxiv2021

Proteins mediating different DNA topologies block RNAP elongation with different efficiency

DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA carrying specific binding sites, while the phage {lambda} CI repressor binds in units of dimers to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by E. coli RNA polymerase through these protein complexes. 186 CI or {lambda} CI bound along unlooped DNA negligibly interfered with transcription by RNAP. More complex topologies induced by scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with rather high affinity, these repressors are not effective roadblocks to transcription.

biophysics↗

bioRxiv2021

A thermodynamic model of bacterial transcription

Transcriptional pausing is highly regulated by the template DNA and nascent transcript sequences. Here, we propose a thermodynamic model of transcriptional pausing, based on the thermal energy of transcription bubbles and nascent RNA structures, to describe the kinetics of the reaction pathways between active translocation, intermediate, backtracked, and hairpin-stabilized pauses. The model readily predicts experimentally detected pauses in high-resolution optical tweezers measurements of transcription. Unlike other models, it also predicts the effect of tension and the GreA transcription factor on pausing.

biophysics↗

bioRxiv2021

Ionic strength modulates HU protein-induced DNA supercoiling

The histone-like protein from E. coli strain U93 (HU) is an abundant nucleoid-associated protein that contributes to the compaction of the bacterial genome as well as to the regulation of many of its transactions. Despite many years of investigations, the way and extent to which HU binding alters the DNA double helix and/or generates hierarchical structures using DNA as a scaffold is not completely understood. Here we combined single-molecule magnetic measurements with circular dichroism studies to monitor structural changes in the DNA-HU fiber as HU concentration was increased from 0 to 1000 nM under low and physiological monovalent salt conditions. We confirmed that DNA compaction correlated with HU concentration in a biphasic manner but DNA unwinding varied monotonically with HU concentration in 100 mM KCl. Instead, in more physiological 200 mM salt conditions, DNA compaction was monotonic while HU-induced DNA unwinding was negligible. Differential compaction and unwinding of DNA may be part of the response of bacteria to large variations in salt concentrations.

biophysics↗

bioRxiv2021

Transcription elongation through lac repressor-mediated DNA loops

Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible. In support of this, RNA polymerase transcribed a looped segment more slowly than an unlooped segment and paused more briefly at LacI obstacles on positively supercoiled templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase appears to facilitate elongation along topologically-complex, protein-coated templates.

biophysics↗

bioRxiv2021

Negative DNA supercoiling tunes protein-mediated looping

Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0-100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0-100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only twelve minutes of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity.

biophysics↗

bioRxiv2021

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. Since the particular sequence of a DNA segment influences both base stacking and hydrogen bonding, the conformational dynamics of B-to-L transitions should be tunable. To test this idea, DNA with diaminopurine replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded A:T base pairs. Triple hydrogen bonding stiffened the DNA by 30% flexurally. In addition, DAP-substituted DNA formed plectonemes with larger gyres for both B- and L-form helices. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing by DNA stretched and unwound by enzymatic activity. The intramolecular nature and ease of this transition likely prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length indicating it is much more contractile and prone to sharp bends and kinks. However, left-handed DAP DNA was twice as stiff as unmodified L-DNA. Thus, significantly doubly and triply hydrogen bonded segments have very distinct mechanical dynamics at physiological levels of negative supercoiling and tension.

biophysics↗