Biology subjects

Frame, L.

Publications and source records attributed to Frame, L..

6 recordsLinked to original sources

bioRxiv2026

Oncogenic ESR1 fusions form chromatin-anchored condensates that remodel nuclear architecture

Estrogen receptor (ESR1) fusions are recurrent drivers of endocrine-resistant breast cancer, yet their mechanisms of action remain poorly understood. Here, we show that ESR1-e6 fusion proteins, including ESR1-DAB2, ESR1-SOX9, and ESR1-YAP1, assemble into nuclear condensates across multiple cell types. Sequence analysis reveals that these fusions combine chromatin-targeting domains with increased intrinsic disorder, promoting multivalent interactions and phase separation. Live-cell imaging, fluorescence recovery after photobleaching, and mechanical perturbation demonstrate that condensates exhibit liquid-like properties, but progressively mature into stable, viscoelastic assemblies constrained by chromatin. Disruption of DNA binding abolishes condensate formation, indicating that chromatin engagement is essential for their assembly and organisation. ESR1 fusion condensates localise to chromatin-depleted regions, reduce nuclear stiffness, and promote chromatin decompaction, consistent with active remodelling of nuclear architecture. We propose that ESR1 fusions exploit phase separation to generate chromatin-anchored assemblies that reorganise the nuclear microenvironment, providing a physical framework through which ESR1 fusions may support sustained transcriptional activity, endocrine resistance and tumour progression.

cell biology↗

bioRxiv2026

DNA gyrase in live bacteria forms liquid condensates through weak multivalent bonding of excess GyrB

Type IIA bacterial topoisomerase DNA gyrase, a GyrA/GyrB heterotetramer, has crucial roles maintaining transcription and DNA replication by relaxing positive DNA supercoils through introducing negative supercoils. However, rates of gyrase-catalysed supercoiling in vitro cannot explain much higher rates required in vivo. To address this puzzle, we used high-speed single-molecule fluorescence imaging of GyrA/GyrB reporters in live Escherichia coli, indicating that cells contain [~]40% more GyrB than GyrA expressed in a diffuse pool or in clusters whose mobility depends on whether they are bound to DNA. Unexpectedly, we discovered that clusters are non-stoichiometric containing [~]150% more GyrB than GyrA, significantly greater than the cellular average, with fluorescence recovery after photobleaching revealing that clustered GyrA and GyrB behave as a liquid whose abundance can be increased by applying gyrase-targeting antibiotics. Structural docking indicates that the liquid state is stabilised through excess GyrB progressively binding to existing clusters via weak, multivalent interactions. By operating in liquid condensates, A2B2 that dissociates from DNA can rebind rapidly instead of diffusing away, increasing enzyme processivity to enable multiple rounds of catalysis that can keep pace with transcription and DNA replication in vivo. This demonstrates a new role for condensates of overcoming kinetic limitations imposed by diffusion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/744598v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1514d4aorg.highwire.dtl.DTLVardef@a95ceeorg.highwire.dtl.DTLVardef@11417daorg.highwire.dtl.DTLVardef@2a2856_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Sticker number modulates pyrenoid condensate assembly to support algal fitness

The valency of intrinsically disordered proteins underpins liquid-liquid phase separation (LLPS), yet how this parameter shapes condensate function and cellular fitness remains poorly understood. Here we exploit the algal pyrenoid-a minimal, two component LLPS system-to directly link condensate properties to physiological performance. Pyrenoid assembly is driven by a disordered, multivalent Linker protein that binds Rubisco at symmetry-related surface sites, with the number of binding motifs ("stickers") varying across species. Using Chlamydomonas reinhardtii, we systematically tuned sticker number from two to nine and examined effects on Rubisco condensation, pyrenoid architecture and CO2 fixation. Three stickers were sufficient for condensation in vitro, but at least four were required for pyrenoid assembly in vivo. Cryo-electron tomography and single-molecule tracking revealed that increasing sticker number enhances Rubisco packing and mobility, while time-resolved imaging and competition assays demonstrated that sticker number governs the kinetics of pyrenoid formation and determines cellular fitness under fluctuating carbon conditions. Our findings establish sticker number as an evolutionary tuning parameter that balances condensate formation, dynamics, and function, providing a quantitative framework for linking the molecular grammar of phase separation to biological fitness.

cell biology↗

bioRxiv2025

Use of a cytochrome P450 humanised mouse model to refine schistosomiasis drug discovery.

Control of schistosomiasis, a neglected tropical disease caused by infection with Schistosoma spp., remains reliant on a single chemotherapy, praziquantel (PZQ). This strategy presents a risk to global health should PZQ-resistant schistosomes establish in endemic areas and justifies the search for new drugs. However, species-specific metabolic differences between humans and preclinical models hinder the optimisation of next-generation anti-schistosomal therapeutics. Here, to bypass these species-specific limitations, we exploited a humanised mouse model, 8HUM, engineered to express the principal human Phase I cytochrome P450 enzymes (CYP1A1/2, CYP2C9, CYP2D6, CYP3A4/7) as well as the transcription factors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in place of 35 murine orthologues. We characterised S. mansoni development, immunopathology, hepatic transcriptomic responses, intestinal microbiome changes and PZQ metabolism as well as PZQ efficacy in 8HUM versus wild-type (WT) mice. 8HUM mice supported normal S. mansoni maturation, infection-associated microbiome dysbiosis, Th2-dominant immune responses and characteristic hepatic pathology. PZQ intrinsic clearance in 8HUM hepatic microsomes mirrored human levels and was >10-fold lower than that found for WT microsomes. Oral dosing revealed human-like PZQ exposures of (R)-PZQ and 4OH-PZQ in 8HUM mice at 25 mg/kg bodyweight and >90% reductions in worm burdens at 100 mg/kg bodyweight (equivalent to that seen in WT mice administered PZQ at 400 mg/kg bodyweight). Our results revealed that 8HUM mice recapitulate key features of murine schistosomiasis while exhibiting human-relevant drug metabolism. These findings establish 8HUM as a refined translational platform for anti-schistosomal drug development, improving predictive accuracy and accelerating therapeutic discovery. One Sentence SummaryA cytochrome P450 humanised mouse model is used to study Schistosoma mansoni development, schistosomiasis, drug metabolism and drug efficacy.

pharmacology and toxicology↗

bioRxiv2021

RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity

The RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA. RecBCD is a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks. It also facilitates coating of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA or RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA crosslinker mitomycin C, we induce DNA damage and quantify the resulting steady state changes in stoichiometry, cellular protein copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA stoichiometries equivalent to several hundred molecules that assemble largely in dimeric subunits before DNA damage, but form periodic subunits of approximately 3-4 molecules within mature filaments of several thousand molecules. Unexpectedly, we find that the physiologically predominant forms of RecB are not only rapidly diffusing monomers, but slowly diffusing dimers.

biophysics↗

bioRxiv2021

Investigating molecular crowding during cell division in budding yeast with FRET

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Forster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

biophysics↗