Biology subjects

Toseland, C. P.

Publications and source records attributed to Toseland, C. P..

13 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

Nuclear Myosin VI stabilises Ku-associated DNA ends during non-homologous end joining

DNA double-strand breaks (DSBs) require rapid signalling and physical stabilisation of broken DNA ends to preserve genome integrity. Here, we identify myosin VI (MVI) as an ATM-regulated component of the DSB response. DNA damage induces rapid nuclear accumulation and nanoscale reorganisation of MVI across multiple cell models, in an ATM-dependent manner. Pharmacological or genetic perturbation of MVI attenuates {gamma}H2AX signalling and disrupts Ku80 organisation, while DNA damage persists. This leads to increased sensitivity to cisplatin and bleomycin. Super-resolution imaging reveals spatial association of MVI with Ku80-containing repair structures, implicating MVI in non-homologous end joining (NHEJ). In a minimal reconstituted system, MVI and actin enhance the proximity of Ku70/80-bound DNA ends. Together, our findings identify MVI as a regulator of DSB repair that links ATM signalling to Ku-associated DNA-end stabilisation and suggest that targeting MVI may sensitise tumour cells to genotoxic therapy.

cancer biology↗

bioRxiv2026

Fluorescence cross-correlation spectroscopy quantifies affinity, cooperativity, and kinetic stability in ternary protein complexes

Many biological processes and emerging therapeutic modalities rely on higher-order protein complexes whose properties cannot be predicted from their constituent binary interactions. However, methods for directly quantifying affinity, cooperativity, and kinetic stability within such assemblies remain limited. Here, we establish fluorescence cross-correlation spectroscopy (FCCS) as a solution-phase approach for characterizing multicomponent protein interactions and apply it to the clinically important HER2-targeting antibodies trastuzumab and pertuzumab. Using fluorescently labelled HER2, trastuzumab, and pertuzumab, we quantified binary binding affinities, directly measured ternary complex formation, and characterized the dissociation kinetics of binary and ternary complexes. FCCS measurements revealed positive cooperativity in the formation of the HER2-trastuzumab-pertuzumab ternary complex, while dissociation experiments demonstrated that the ternary complex is kinetically more stable than the corresponding binary interactions. Together, these findings provide direct solution-phase evidence that cooperative interactions stabilize the HER2-trastuzumab-pertuzumab complex, offering a molecular explanation for the enhanced efficacy of dual HER2 targeting in cancer therapy. More broadly, this work demonstrates that FCCS can robustly quantify affinity, cooperativity, and kinetic stability of multicomponent protein complexes using a commercially available platform. We provide a broadly accessible framework for studying higher-order protein interactions and supporting the development of next-generation multispecific and combination therapeutics. Significance StatementMany proteins function as part of multicomponent complexes, yet most experimental methods characterize interactions only one pair at a time, or indirectly through secondary reporters, or necessitating saturation of binary interactions first. We show that fluorescence cross-correlation spectroscopy can directly quantify how multiple binding partners interact simultaneously by measuring affinity, cooperativity, and kinetic stability in solution. Applying this approach to the clinically important HER2-targeting antibodies trastuzumab and pertuzumab reveals cooperative stabilization of their ternary complex. Because the measurements are performed on a commercially available instrument and require no surface immobilization, this broadly accessible method should facilitate mechanistic studies of complex biomolecular interactions and aid the development of multispecific and combination therapeutics.

biophysics↗

bioRxiv2026

DNA Damage Driven Viability Loss and Transcriptional Reprogramming in Chinese Hamster Ovary Cell Perfusion Culture

Intensified perfusion cultures promise higher yields and consistent product quality, yet extended runs frequently stall due to declining Chinese hamster ovary (CHO) cell viability at high cell density. Here, we identify DNA damage accumulation as a central, previously underappreciated driver of this limitation. Using two antibody-expressing CHO cell lines operated at a control and high cell density in perfusion bioreactors, we combined time-resolved transcriptomics with molecular, biophysical and super-resolution imaging analyses. We observed a progressive, global downregulation of DNA damage response (DDR) pathways accompanied by a time-dependent accumulation of DNA lesions. Notably, {gamma}H2AX signalling declined despite DNA damage, indicating impaired damage sensing and repair. Concomitantly, RNA polymerase II protein levels and transcriptional hub organisation were markedly reduced, consistent with widespread transcriptional dysfunction preceding loss of viability. An extended 21-day perfusion run confirmed continued viability decline beyond day 14, supporting a cumulative damage model. Comparison with HEK293 cells revealed substantially more efficient DNA repair, underscoring an intrinsically attenuated DDR in CHO cells. Together, these findings demonstrate that unrepaired DNA damage fundamentally constrains perfusion culture longevity, exposing limits of CHO genomic plasticity and highlighting DDR pathways as promising targets for host cell engineering to enhance perfusion performance.

bioengineering↗

bioRxiv2025

Myosin VI orchestrates estrogen-driven gene expression in breast cancer cells.

Approximately 75% of breast cancer cases are estrogen receptor (ER)-positive, with endocrine therapies forming the foundation of treatment. However, therapeutic resistance remains a major clinical challenge, necessitating the identification of new molecular targets. Myosin VI (MVI), a motor protein increasingly linked to cancer, directly interacts with the ER and plays a key role in the spatial regulation of RNA Polymerase II. Notably, expression levels of MVI and ER are positively correlated across breast cancer tissues. Here, we present a multidisciplinary investigation combining advanced imaging, genomic profiling, and phenotypic characterisation to elucidate the interplay between MVI and the ER. We demonstrate that MVI nuclear localisation is dynamically regulated by estrogen signalling and ER activity. Conversely, ER nuclear localisation requires active MVI, suggesting a reciprocal regulatory mechanism. Furthermore, MVI influences subnuclear architecture, modulating ER transcriptional activity and downstream gene expression programmes that govern cell proliferation and migration. Importantly, pharmacological inhibition of MVI enhances the effect of hormone therapy, resulting in greater disruption of ER function than monotherapy alone. Moreover, MVI inhibition also suppresses the activity of hormone-resistant ER mutants, highlighting its potential to overcome therapy resistance. Our findings establish MVI as a critical regulator of ER nuclear dynamics and gene expression, supporting its candidacy as a novel therapeutic target in ER-positive breast cancer.

cancer biology↗

bioRxiv2023

3D environment modulates persistent lamin distribution and the biomechanical signature of the nucleus.

The interplay between cells and their surrounding microenvironment drives multiple cellular functions, including migration, proliferation, and cell fate transitions. The nucleus is a mechanosensitive organelle that adapts external mechanical and biochemical signals provided by the environment into nuclear changes with functional consequences for cell biology. However, the morphological and functional changes of the nucleus induced by 3D extracellular signals remain unclear. Here, we demonstrated that cells derived from 3D conditions conserve changes from cell confinement and show an aberrant nuclear morphology and localization of lamin B1, even in the absence of cellular confinement. We found that actin polymerization and protein kinase C (PKC) activity mediate the abnormal distribution of lamin B1 in 3D conditions-derived cells. These cells present altered chromatin compaction, gene transcription and cellular functions such as cell viability and migration. By combining biomechanical techniques and single-nucleus analysis, we have determined that the nucleus from 3D conditions-derived cells shows a different mechanical behavior and biophysical signature than the nucleus from control cells. Together, our work substantiates novel insights into how the extracellular environment alters the cell biology by promoting permanent changes in the chromatin, morphology, lamin B1 distribution, and the mechanical response of the nucleus.

cell biology↗

bioRxiv2022

Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription

NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

biophysics↗

bioRxiv2021

A targeted and tuneable DNA damage tool using CRISPR/Cas9.

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-Strand Breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR associated protein 9) endonuclease enzyme, is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

cell biology↗

bioRxiv2020

Binding partners regulate unfolding of myosin VI to activate the molecular motor

Myosin VI is the only minus-end actin motor and is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single molecule imaging to investigate the impact of binding partners upon myosin VI molecular organisation and dynamics.

biophysics↗

bioRxiv2020

High throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

Mechanobiology is focused on how the physical forces and the mechanical properties of proteins, cells and tissues contribute to physiology and disease. While the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. The broad applicability and versatility of our approach has been demonstrated through in vitro force-induced enzymatic activity and conformation changes, along with force-induced receptor activation and their downstream signalling pathways in live cells. Overall, our methodology allows for the first-time ensemble biochemical and cell-based assays to be performed under force, in high throughput format. This novel approach would substantially add to the mechano-biological toolbox and increase the availability of mechanobiology measurements.

biophysics↗

bioRxiv2020

Nuclear myosin VI regulates the spatial organization of mammalian transcription initiation.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability which underpin the function these transcription factories remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI in the nanoscale organisation of RNAPII. We reveal that myosin VI acts as the molecular anchor that holds RNAPII into transcription factories. Perturbation of myosin VI leads to the disruption of RNAPII localisation, changes in chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of myosin VI in the spatial regulation of gene expression during the rapid response to changes in the cellular environment.

cell biology↗

bioRxiv2019

Binding partner regulation of Myosin VI: Loss of tumour-suppressor Dab2 leads to enhanced activity of nuclear myosin

Myosin VI is involved in a variety of cellular processes ranging from endocytosis to transcription. This multi-functional potential is achieved through alternative isoform splicing and through the interaction with a diverse network of binding partners. However, the interplay between the two modes of regulation remains unexplored. To this end, we have compared two different binding partners, Dab2 and CALCOCO2/NDP52, and their interaction with two myosin VI splice isoforms. We found that both isoforms adopt an auto-inhibited state and are subsequently activated by binding partner association. However, differential regulation is achieved through a high and a low affinity binding motifs within myosin VI, with one isoform having the high affinity site blocked. This allows competition between partners and links isoform splicing with binding partner selectivity. Dab2 competition hinders the activity of nuclear myosin VI by preventing DNA binding and transcription. Moreover, re-introduction of Dab2 in the Dab2-deficient MCF-7 cells leads to a decrease in myosin VI-dependent estrogen receptor gene expression. We propose that the frequent loss of Dab2 during the onset of cancer enables a higher level of nuclear myosin VI activity, thereby driving the activity of the estrogen receptor to promote tumourgenesis.

cell biology↗

bioRxiv2017

A Reagentless Biosensor For mRNA: A New Tool To Study Transcription

Gene expression, catalysed by RNA polymerases, is one of the most fundamental processes in living cells. Yet, the means to study their activity are currently limited. The majority of methods to quantify mRNA are based upon initial purification of the nucleic acid. This leads to experimental inaccuracies and loss of product. Here, we describe the use of a reagentless mRNA fluorescent biosensor based upon the single stranded binding (SSB) protein. In this study, SSB showed similar binding properties to mRNA, to that of its native substrate, ssDNA. Furthermore, fluorescently labelled MDCC-SSB gave the same fluorescence response with both ssDNA and ssRNA, in a concentration dependent manner. When directly compared to RT-qPCR, we found the biosensor to be more reproducible with no product lost through purification. Therefore, the MDCC-SSB is a novel tool for comparative measurement of mRNA yield following in vitro transcription.

biochemistry↗