Biology subjects

Rico, F.

Publications and source records attributed to Rico, F..

15 recordsLinked to original sources

bioRxiv2026

Scaling the Dynamics of Coiled Coils

Coiled coils are structural motifs in proteins that play diverse functions. In MRE11-RAD50 (MR) complexes, ATP-driven changes in coiled coils are essential for DNA break sensing. However, coiled coil dynamics and its modulation by protein conformational changes remain unclear, partly due to the lack of quantitative tools. Here, we used high-speed atomic force microscopy (HS-AFM) for real-time visualization of the coiled coil conformational dynamics of individual MR complexes from bacteria and human homologs, and a biomedically relevant variant. The mean square deviation of the end-to-end distance of the coiled coils revealed a power-law scaling with time, conserved across conformational states, homologs, and variants, suggesting a universal dynamic scaling. Coiled coils behave as semi-flexible filaments with strong internal friction, leading to relaxation times that were seconds-long and varied among conformational states and variants. Molecular dynamics simulations indicated that strong friction arose from long-lifetime contacts between coils. Our results suggest that MR complexes modulate the coiled coil dynamics to mediate long-range allosteric and allodynamic communication during DNA repair.

biophysics↗

bioRxiv2026

EpCAM sustains crypt mechanics and prevents aberrant remodeling

Crypt morphogenesis is a fundamental process that drives intestinal growth and maintains its homeostasis. Although intestinal tissue mechanics is recognized as pivotal for early crypt initiation and budding, the molecular mechanisms that couple epithelial forces to mature crypt behavior remain largely unexplored. In particular, while Paneth cells have been proposed to be implicated in the mechanical control of crypt remodeling and fission, the underlying pathways have remained elusive. Here, we identified EpCAM (Epithelial Cell Adhesion Molecule) as a key regulator of epithelial contractility and crypt morphogenesis. EpCAM was preferentially enriched at lateral interfaces of Paneth cells in vivo and in organoid models, where it maintained cortical tension and contractile organization. Loss of EpCAM disrupted crypt cell cortical myosin II-A localization, leading to aberrant Paneth cell apical contractility, enlarged apical surfaces, distorted pyramidal morphology, and altered aspect ratios. Consequently, Paneth cells lose their tight clustering in crypt base, their spatial distribution along the crypt axis becomes irregular, and the collective architecture of the crypt epithelium was compromised. At the tissue level, EpCAM deficiency profoundly perturbed crypt fission, increasing fission frequency while favoring asymmetric outcome both in EPCAM-mutated Congenital Tufting Enteropathy (CTE) patients. Live imaging of EpCAM-knockout mouse organoids confirmed a failure in the spatial control of crypt bifurcation. Collectively, our findings revealed that EpCAM is not required to initiate fission, but is essential to coordinate the mechanical forces and ensure correct division of the crypt. We showed that EpCAM-dependent biomechanical integrity of Paneth cells generates a stabilizing force field at the crypt base, orchestrating coordinated tissue remodeling. Disruption of this program led to unbalanced force transmission, distorted crypt geometry, and aberrant fission outcomes, revealing a critical link between epithelial mechanics and intestinal morphogenesis.

developmental biology↗

bioRxiv2026

A non-invasive approach for understanding localized force generation in 3D tissues

The development, maintenance and repair of epithelial tissues critically rely on adhesion complexes that ensure structural integrity while enabling dynamic remodeling. Such tissue remodeling underpins both physiological morphogenesis and pathological transformation. Central to these processes are mechanical forces, which tightly couple cytoskeletal organization to adhesion dynamics. Despite extensive investigations in two-dimensional (2D) systems, how these interactions are orchestrated within polarized three-dimensional (3D) epithelia remains largely unresolved. Here, we introduce a new, non-invasive strategy to probe localized force generation within 3D epithelial tissues. We engineered elastic polyacrylamide (PAAm) microbeads with cell-mimetic size and mechanical properties, enabling their seamless integration. In contrast to conventional bead injection approaches, these PAAm microbeads were spontaneously engulfed by the tissue, thereby establishing an intrinsic interface through which bead deformation can be directly correlated with local cytoskeletal architecture and adhesion organization, as visualized through high-resolution imaging combined with quantitative 3D computational reconstruction. Using this approach, we demonstrated that localized mechanical perturbations trigger pronounced cytoskeletal remodelling while preserving global tissue polarity. We further identified the extracellular matrix composition as key determinant of bead-tissue interactions, with collagen-I coating promoting robust adhesion and efficient incorporation. At the bead-cell interface, cells assembled tension-bearing focal adhesions and organized actin stress fibers, revealing the emergence of active cortical stress. Strikingly, quantitative analysis of bead deformation revealed a previously unrecognized mechanical duality: spatially segregated regions of pulling and pushing forces coexisted at the microscale, directly correlated with local cytoskeleton dynamics. This finding challenges the prevailing view of homogenous force application and instead supports a model in which cells deploy highly coordinated and spatially patterned force-generating strategies. Altogether, this integrative and non-invasive strategy offers a comprehensive pipeline for dissecting the dynamic interplay between cellular processes and tissue mechanics during morphogenesis in 3D model systems.

cell biology↗

bioRxiv2026

PySteMoDA: An Open Source Python Package for the Analysis of Steered Molecular Dynamics Simulations Data

Molecular dynamics simulation is a powerful computational technique used for predicting and understanding the dynamic behavior of biomolecular systems. Steered molecular dynamics (SMD) simulations enable the study of force-induced processes in biomolecules, effectively mimicking single-molecule force spectroscopy experiments probing protein unfolding and receptor-ligand unbinding. Given the stochastic nature of these mechanical events, accurately exploring the dynamic behavior of biomolecules and extracting accurate physical information requires several in-silico experiments. This includes performing many pulling simulations at different velocities or force loading rates. The large amount of data obtained from these simulation sets requires efficient automated data processing tools. We present PySteMoDA, a novel Python package with a user-friendly graphical interface specifically designed for constant-velocity SMD data analysis. The automated force peak detection methods reduce user bias, improve accuracy, and accelerate data analysis. The package also allows identification of residues involved in mechanical events through computation of the time-dependent mechanical work and correlation factors between residue pairs. This package not only addresses automated data processing in SMD simulations and accurate parameter extraction, but also significantly enhances accessibility and usability. Through PySteMoDA, users can efficiently analyze simulation data without the barrier of coding, facilitating a wider range of investigations and insights in the field of computational biochemistry and biophysics.

biophysics↗

bioRxiv2025

Near equilibrium unbinding of streptavidin/biotin using single molecule acoustic force spectroscopy.

The dissociation of the streptavidin-biotin (SA-b) bond has been widely characterized using bulk and single molecule force spectroscopy (SMFS) techniques. However, the dissociation rates (koff) from SMFS ([~]10-1 s-1) typically do not align with those from bulk approaches ([~]10-6 - 10-5 s-1), likely because SMFS measurements are conducted far from equilibrium. Near equilibrium SMFS requires high throughput measurements to obtain large enough statistics, and high stability over long time measurements at ultraslow loading force rates, impractical in most SMFS techniques. Here, we developed in-situ force calibration strategies for acoustic force spectroscopy (AFS) to probe the unbinding forces of SA-b in the near equilibrium regime, from 10 pN/s down to 10- 3 pN/s. The resulting koff was in excellent agreement with that from bulk measurements on the very same system. Combined with our previous SA-b data, we covered 15 orders of magnitude in loading rate, setting the ground for SMFS over the widest dynamic range, essential to fully describe the energy landscape of biomolecular processes.

biophysics↗

bioRxiv2025

Real-time Visualization of Trigger Factor on Translating Ribosomes

Trigger Factor (TF) is an ATP-independent chaperone that assists in co-translational protein folding by associating with ribosomes to prevent aggregation. While its interaction with ribosomes has been described, real-time visualization of TF dynamics has remained elusive. Using high-speed atomic force microscopy, we imaged full 70S ribosomes under near-physiological conditions during translation. TF exhibited dynamic transitions between extended and compact conformations, forming both stable and transient contacts with ribosomal proteins uL23 and bL17 in ribosome-nascent chain complexes. Binding to non-translating ribosomes was not observed under these conditions. Molecular dynamics simulations of TF alone and in complex with ribosomal proteins supported the experimental observations. Our findings reveal the structural flexibility of TF and its selective association with active ribosomes. Our combination of experimental and computational approaches offers new insights into how TF dynamically engages ribosomes during translation to facilitate protein folding.

molecular biology↗

bioRxiv2024

Modulation of SARS-CoV-2 spike binding to ACE2 throughconformational selection

The first step of SARS-CoV-2 infection involves the interaction between the trimeric viral spike protein (S) and the host angiotensin-converting enzyme 2 (ACE2). The receptor binding domain (RBD) of S adopts two conformations: open and closed, respectively, accessible and inaccessible to ACE2. Therefore, RBD motions are suspected to affect ACE2 binding; yet a quantitative description of the underlying mechanism has been elusive. Here, using single-molecule approaches, we visualize RBD opening and closing and probe the S/ACE2 interaction. Our results show that RBD dynamics affect ACE2 binding but not unbinding. The resulting modulation is quantitatively predicted by a conformational selection model in which each protomer behaves independently. Our work reveals a general molecular mechanism affecting binding affinity without altering binding strength, helping to understand coronavirus infection and immune evasion.

biophysics↗

bioRxiv2023

MechanoProDB: A Web Based Database for Exploring the Mechanical Properties of Proteins

The mechanical stability of proteins is crucial for biological processes. To understand the mechanical functions of proteins, it is important to know the protein structure and mechanical properties. Protein mechanics is usually investigated through force spectroscopy experiments and simulations that probe the forces required to unfold the protein of interest. While there is a wealth of data in the literature on force spectroscopy experiments and steered molecular dynamics simulations of forced protein unfolding, this information is spread and difficult to access by non-experts. Here we introduce MechanoProDB, a novel web-based database resource for collecting and mining data obtained from experimental and computational works. MechanoProDB provides a curated repository for a wide range of proteins, including muscle proteins, adhesion molecules and membrane proteins. The database incorporates relevant parameters that provide insights into the mechanical stability of proteins and their conformational stability such as the unfolding forces, energy landscape parameters and contour lengths of unfolding steps. Additionally, it provides intuitive annotations of the unfolding pathways of each protein, allowing users to explore the individual steps during mechanical unfolding. The user-friendly interface of MechanoProBD allows researchers to efficiently navigate, search and download data pertaining to specific protein folds or experimental conditions. Users can visualize protein structures using interactive tools integrated within the database, such as Mol*, and plot available data through integrated plotting tools. To ensure data quality and reliability, we have carefully manually verified and curated the data currently available on MechanoProDB. Furthermore, the database also features an interface that enables users to contribute new data and annotations, promoting community-driven comprehensiveness. The freely available MechanoProDB aims to streamline and accelerate research in the field of mechanobiology and biophysics by offering a unique platform for data sharing and analysis. MechanoProDB is freely available at https://mechanoprodb.ibdm.univ-amu.fr.

biophysics↗

bioRxiv2023

Reliable, standardized measurements for cell mechanical properties

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Youngs modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

biophysics↗

bioRxiv2022

Coupled mechanical mapping and interference contrast microscopy reveal viscoelastic and adhesion hallmarks of monocytes differentiation into macrophages

Monocytes in the blood torrent, when activated by pro-inflammatory signals, adhere to the vascular endothelium and migrate into the tissue for ultimately differentiate into macrophages. Mechanics and adhesion play a crucial role in macrophage functions, such as migration and phagocytosis. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we combined atomic force microscopy (AFM) viscoelastic mapping with interference contrast microscopy (ICM) to simultaneously probe, at the single-cell level, viscoelasticity and adhesion during monocyte differentiation. THP-1 monocytic cells were differentiated into macrophages through phorbol 12-myristate 13-acetate (PMA). Morphological quantification was achieved using holographic tomography imaging and the expression of integrin subunit CD11b was tracked as a marker of differentiation. Holographic tomography proved to be a quantitative in vivo technique, revealing a dramatic increase in macrophage volume and surface area and two subpopulations, spread and round macrophages. AFM viscoelastic mapping revealed an increased stiffness and more solid-like behavior of differentiated macrophages, especially in the lamellipodia and microvilli regions. Differentiated cells revealed an important increase of the apparent Youngs modulus (E0) and a decrease of cell fluidity ({beta}) on differentiated cells, which correlated with an increase in adhesion area. Macrophages with a spreading phenotype enhanced these changes. Remarkably, when adhesion was eliminated, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activity, such as phagocytosis, making it more efficient. Our proposed approach revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

biophysics↗

bioRxiv2022

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

Single-molecule data are of great significance in biology, chemistry, and medicine. However, experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are needed. Acoustic force spectroscopy (AFS) is an emerging manipulation technique which generates acoustic waves to apply force in parallel on a large population of microbeads tethered to a surface. We have exploited this configuration on a recently developed modular Junctured-DNA (J-DNA) scaffold designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measured its unbinding kinetics under force at the single-bond level. Special effort was made in analyzing the data in order to identify potential pitfalls. We established a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy for measuring the force dependent rupture of a single domain antibody with its antigen. We get a good agreement with standard measurement at zero force. Our technique offers single molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

biophysics↗

bioRxiv2022

BIN1/Amphiphysin 2 and ezrin drive filopodia-like structures in myoblasts

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

cell biology↗

bioRxiv2022

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.

cell biology↗

bioRxiv2022

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 {micro}m wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe1 that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

biophysics↗

bioRxiv2018

Monitoring Unfolding of Titin I27 single- and bi-Domain with High-Pressure NMR Spectroscopy.

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular Dynamics (MD) has been associated often to force spectroscopy experiments to decipher the unfolding mechanism of titin Ig-like single- or multi-domain, the giant multi-modular protein from sarcomere, yielding information on the sequential events during titin unfolding under stretching. Here, we used high-pressure NMR to monitor the unfolding of titin I27 Ig-like single-domain and tandem. Since this method brings residue-specific information on the folding process, it can provide quasi-atomic details on this process, without the help of MD simulations. Globally, the results of our high-pressure analysis are in agreement with previous results obtained by the association of experimental measurements and MD simulation and/or protein engineering, although the intermediate folding state caused by the early detachment of the AB {beta}-sheet, often reported in previous works based on MD or force spectroscopy, cannot be detected. On the other hand, the AG parallel {beta}-sheet of the {beta}-sandwich has been confirmed as the Achilles heel of the 3D scaffold: its disruption yields complete unfolding, with very similar characteristics (free energy, unfolding volume, kinetics constant rates) for the two constructs.

biophysics↗