Biology subjects

Marie, R.

Publications and source records attributed to Marie, R..

7 recordsLinked to original sources

bioRxiv2026

SurpHer: a genetically encoded ratiometric sensor for dynamic extracellular pH imaging

Extracellular pH is a key microenvironmental factor shaping cell physiology and disease, creating a need for quantitative biosensors that can capture dynamic changes in pHe at the surface of individual living cells. Here, we develop a genetically encoded, ratiometric extracellular pH biosensor through systematic screening of a modular library of membrane-display designs that combine SEpHluorin with a pH-stable reference fluorophore. Screening identified a cell-surface-localised mKate2-SEpHluorin construct, named SurpHer, that exhibits dynamic ratiometric responses across the pHe range of 6 - 7.8. SurpHer shows robust membrane localisation and extracellular pH responsiveness across diverse human cell types including HEK293T, PANC-1 and MDA-MB231 cells. Following stable integration in MDA-MB-231 cells, SurpHer enabled time-course imaging of pHe gradients in a microfluidic platform for modelling tumour microenvironments. SurpHer enables real-time interrogation of the pericellular pH environment of tumor cells and, more broadly, provides a strategy to probe microenvironmental pH dynamics across diverse biological contexts.

cell biology↗

bioRxiv2025

Calibration-free molecular counting from a single DNA-PAINT intensity trace using cumulants

Single-molecule localization microscopy achieves nanometer-scale resolution but fails to count molecular targets when multiple targets are in close proximity. DNA-PAINT uses reversible binding of fluorescently labeled probes to image molecular targets, but existing time-series based counting methods require prior knowledge of binding kinetics, calibration, or extensive data post-processing, which limits applicability across heterogeneous biological samples. Here, we present mCOAST, a calibration-free method that extracts molecular counts and kinetic parameters directly from a single DNA-PAINT intensity trace using cumulants. Unlike existing approaches, mCOAST requires no adjustable parameters, data normalization, or denoising. We demonstrate accurate counting for diffraction-limited clusters with up to 48 targets at high imager concentrations, with precision that improves, rather than decreases, as concentration increases. Critically, mCOAST counts targets in individual clusters despite kinetic heterogeneity across samples or between experiments. This paves the way towards quantitative imaging and counting in uncalibrated biological systems, such as living cells.

biophysics↗

bioRxiv2025

A microfluidic model to recapitulate pH and oxygen gradients in solid tumors

The tumor microenvironment (TME) plays critical roles in cancer development, aggressiveness, and treatment resistance. The TME comprises cellular (stromal) components as well as gradients of physicochemical properties, including hypoxia and acidosis. Understanding of how hypoxia and acidosis gradients impact cancer phenotypes is lacking, in large part due to challenges in precisely mimicking and controlling such gradients in a manner compatible with the growth of cancer- and stromal cells. Here, we design and validate a microfluidic device enabling orthogonal gradients of oxygen and pH. Both gradients are established by diffusion from a nearby source and sink in the observation area in the absence of flow. This produces linear gradients at steady state. Our device enables a wide range of spatiotemporally resolved analyses, from omics to live cell imaging, interrogating the impact of the physicochemical TME on disease development. The design is easily adaptable, making it valuable for a wide range of questions involving physicochemical gradients.

cancer biology↗

bioRxiv2025

In-solution Characterization of Biomolecular Interaction Kinetics under Native Conditions

Characterizing the kinetics of biomolecular interactions is fundamental for understanding biological mechanisms, devel-oping novel drugs for advancing healthcare and for optimizing processes in protein engineering. Although modern sur-face-based methods have advanced our understanding of protein-protein and protein-ligand kinetics, they rely on immobilized samples, preventing the study of interactions under native conditions and leading to an incomplete understanding. In this work, we propose a paradigm shift by introducing a new method based on flow-induced dispersion analysis to study interaction kinetics while keeping biomolecules in solution, eliminating the need for surface immobilization and thereby preserving molecular mobility and avoiding structural constraints. The method examines reactions outside equilibrium conditions by inducing a rapid concentration change in one of the binding partners (C-Jump) in a controlled microfluidic environment. Notably, it operates without buffer restrictions and requires only minimal sample quantities. We demonstrate C-Jumps capability by accurately determining the association and dissociation rates of both protein-protein and protein-small molecule interactions. Furthermore, we validate its robustness by measuring the rates of a protein-protein interaction in human serum as well as a protein-small molecule interaction in-solution and label free. This under-lines C-Jumps broad applicability for studying biomolecular interactions under native conditions, offering a powerful tool for advancing protein engineering and drug discovery, as well as enabling the characterization of previously inaccessible interactions. TABLE OF CONTENTThis work presents C-Jump, a breakthrough method for the direct measurement of kinetic rates in solution, closely mimicking physiological conditions. Utilizing flow-induced dispersion analysis, C-Jump determines rate constants with high accuracy for protein-protein and protein- small molecule interactions. Free from buffer constraints and requiring only nanograms of protein, it can operate label-free, transforming biomolecular interaction studies across biophysics, chemistry, and medicine. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/645748v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@11385ceorg.highwire.dtl.DTLVardef@b5d88forg.highwire.dtl.DTLVardef@9cc2cforg.highwire.dtl.DTLVardef@1b9d17c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Continuous Titration Based Method For Rapid In-solution Analysis Of Non-covalent Interactions

Development of new drugs typically involves the identification and validation of molecular inhibitors or promotors of endogenous biological processes. The identification of ligands that can bind the target of interest is typically achieved by screening large libraries of small molecules, using analytical methods that only provide yes/no answers. These methods are only qualitative and often associated with unacceptable amounts of false positives and negatives. Quantitative methods are in general more accurate but time intensive. This is mainly due to repeating measurements of a dilution series in order to generate a titration curve and measure the dissociation constant (Kd). In this work, we introduce Continuous Titration Based Spectral Related Intensity Change (cSPRING), that combines Taylor dispersion analysis (TDA) with ratiometric fluorescence detection to measure a Kd in a single experiment. cSPRING is an in-solution method that reduces the sample preparation time 8-fold and requires only nanograms of protein. We show a good agreement of cSPRING with other quantitative methods for three well-known protein-small molecule interactions with binding affinities ranging from the low nanomolar to high micromolar. In addition, we show that cSPRING is able to measure binding affinities in under a minute, highlighting its efficiency and potential for screening applications.

biophysics↗

bioRxiv2024

AML patient blasts exhibit polarization defect upon interaction with bone marrow stromal cells.

Hematopoietic stem and progenitor cells (HSPCs) establish specific interactions with bone marrow stromal cells, leading to their polarization. Given the role of cell polarity in protection against tumorigenesis and the importance of the niche in hematological disorders such as acute myeloid leukemias (AMLs), we investigated the polarization capacities of leukemic blasts from patients. Using engineered micro-niches and centrosome position with respect to the contact site with stromal cells as a proxy for cell polarization, we showed that AML cell lines and primary cells from AML patient blasts were unable to polarize in contact with healthy stromal cells. In return, exposure to AML patient-derived stromal cells compromised the polarization of healthy adult HSPCs and AML blasts from patients. Using live cell imaging in engineered "bone-marrow-on-a-chip", we further revealed that stromal cells from a leukemic niche increased the migration speed and distance of healthy HSPCs and AML blast as compared to their behavior in contact with healthy stromal cells. The results collectively demonstrated the respective influences of intrinsic AML blast transformation and extrinsic contact with AML stromal cells on the defective polarization of AML blast. They suggested that leukemic progression is associated with cell polarization defects and proposed new methodological approaches to investigate this relationship in AML progression.

cancer biology↗

bioRxiv2018

Sequencing Metrics of Human Genomes Extracted from Single Cancer Cells Individually Isolated in a Valveless Microfluidic Device

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cell (LS174T, LS180 and RKO) lines and fresh colorectal cancers are individually trapped, their genomes extracted and prepared for sequencing, using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 {+/-} 0.06 %) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as extraction is in sub-nanoliter volumes. Our data also demonstrates that high quality single cell sequencing can be achieved using a relatively simple, inexpensive and scalable device.

genomics↗