Biology subjects

Berro, J.

Publications and source records attributed to Berro, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Lipid diffusion enhanced by molecular motors

An experimental study of myosin I motors on a supported lipid membrane with a nematic actin filament layer is presented. The activities of myosin I motors re-organize the actin layer, in particular the morphology of +1/2 nematic defects. Data from single-particle tracking (SPT) of lipid molecules reveal two interesting features. First, the distribution of average lipid speed over a track shows two groups of lipids (high-speed and low-speed), and the proportion of lipids to be high-speed is higher in regions rich in actin. Second, by classifying short-time movement of the lipids into normal diffusion, directed motion, confined motion, and subdiffusion states, we found that lipids in regions rich in actin have a lower probability to stay in the confined and subdiffusion states and more likely to perform normal diffusion. Furthermore, fluorescence recovery after photobleaching (FRAP) measurements show that the diffusion of lipids in the actin-rich regions is enhanced. The SPT and FRAP results suggest that myosin I motor activities turn low-speed lipids to high-speed ones, and affect the lipid moving state by reducing confined and subdiffusive motion. As a result, lipid diffusion is enhanced by motor activities.

biophysics↗

bioRxiv2026

Mechanical contributions of the Myp2 tail revealed by coiled-coil force sensors

Cytokinesis is driven by the constriction of an actomyosin contractile ring, yet how forces generated by individual myosin molecules are transmitted within the ring remains poorly understood. In fission yeast Schizosaccharomyces pombe, the type II myosin Myp2 plays a key role in ring constriction and stability, particularly under stress conditions. Unlike typical type-II myosins, Myp2s tail contains two coiled coils. Although numerous studies have revealed the structure and molecular function of Myp2, the mechanical contribution of its unique tail region has remained unclear. Here, we directly measured forces acting on Myp2 in living cells using genetically encoded coiled-coil force sensors. Insertion of force sensors into the neck region revealed that Myp2 experiences forces of approximately 6.6 pN within the contractile ring. To dissect the contribution of the tail region, we systematically analyzed tail-deletion mutants. Deletion of the proximal tail had little effect on force levels, whereas deletion of the distal C-terminal tail significantly reduced the forces experienced by Myp2. Quantitative analysis of force distributions further showed that truncation of the C-terminal tail reduces the characteristic force by approximately half, demonstrating a major mechanical contribution of the distal tail to the forces experienced by Myp2 at the molecular scale. Deletion of the C-terminal tail slowed the growth rate, consistent with previous macroscopic observations that this region is required for robust ring constriction under stress conditions. We further found that the C-terminal tail forms clusters, which require full-length Myp2 for proper localization to the ring. These findings, together with molecular force measurements and quantitative analysis, demonstrate that the Myp2 tail is not merely a structural or regulatory domain but functions as a load-bearing component in vivo, with its C-terminal region playing a central role in force transmission across scales.

Cell Biology↗

bioRxiv2025

Mechanisms of force transmission on cytokinesis Formin Cdc12 in fission yeast revealed by coiled-coil force sensors

Cytokinesis is a fundamental process in cell division, where an actomyosin contractile ring plays a central role in completing the cell division. Although some experimental and computational efforts have evaluated ring tension and the molecular organization of rings, the mechanisms of force transmission at the molecular level remain unclear. Here, we used our novel coiled-coil force sensors to measure the force distribution along the formin Cdc12, a key cytokinesis protein in fission yeast. Our force measurements revealed that individual formin Cdc12 molecules transmit up to [~]6 pN with distinct mechanisms between the regions upstream and downstream of the formin homology 2 (FH2) domain, which binds the barbed ends of actin filaments. The force transmitted on the N-terminal region before the FH2 domain requires anchoring to a cytokinetic node via the Cdc15 binding region, but is independent of the C-terminal region after FH2. In contrast, the region after the FH2 domain transmits forces independently of the Cdc12s N-terminal region. Force screening using the coiled-coil force sensors found that a region in Cdc12s disordered C-terminal tail can associate with the contractile ring. Altogether, the force measurements with our coiled-coil force sensors allowed us to uncover the mechanisms of force transmission along Cdc12 and characterize new motifs and binding partners.

cell biology↗

bioRxiv2025

Asymmetric phosphoinositide lipid bilayers generated by spontaneous lipid insertion

Phosphatidylinositol phosphate (PIP) lipids, enriched on the cytoplasmic leaflet of the plasma membrane, are key regulators of diverse cellular processes, often through interactions with partner proteins that regulate actin assembly. Supported lipid bilayers (SLBs) provide a powerful model system to study the interactions of PIP lipids with their partner proteins. However, despite advances in SLB preparation methods, it remains a challenge to robustly obtain fluid SLBs in which PIP lipids are both mobile and asymmetrically distributed. In this study, we report a simple and robust method to generate asymmetric SLBs containing tunable amounts of PI(4,5)P2. By dissolving PI(4,5)P2 below its critical micelle concentration (CMC), we enable its spontaneous insertion into the upper leaflet of SLBs exposed to bulk solution. The mobility of PI(4,5)P2 is confirmed using fluorescence recovery after photobleaching (FRAP). Furthermore, we demonstrate that PI(4,5)P2 incorporated using this method retains its functionality, recruiting binding partners, actin-membrane linker ezrin, and myosin 1 motors capable of sliding actin filaments on the SLBs. Our method offers a straightforward strategy to generate asymmetric PI(4,5)P2-containing SLBs and is applicable to other lipid species with high CMC values.

biophysics↗

bioRxiv2025

Genetically encoded mechano-sensors with versatile readouts and compact size

Mechanical forces are critical for virtually all fundamental biological processes, yet quantification of mechanical forces at the molecular scale in vivo remains challenging. Here, we present a new strategy using calibrated coiled coils as genetically encoded, compact, tunable, and modular mechano-sensors to substantially simplify force measurement in vivo, via diverse readouts (luminescence, fluorescence and analytical biochemistry) and instrumentation readily available in biology labs. We demonstrate the broad applicability and ease-of-use of these coiled coil mechano-sensors by measuring forces during cytokinesis (formin Cdc12) and endocytosis (epsin Ent1) in yeast, force distributions in nematode axons ({beta}-spectrin UNC-70), and forces transmitted to the nucleus (mini-nesprin-2G) and within focal adhesions (vinculin) in mammalian cells. We report discoveries in intracellular force transmission that have been elusive to existing tools.

cell biology↗

bioRxiv2024

Nodal Modulator (NOMO) is a force-bearing transmembrane protein required for muscle differentiation

The endoplasmic reticulum (ER) relies on specialized membrane-shaping proteins to maintain a continuous network of sheets and tubules that host distinct biological processes. How this intricate structure of the ER membrane system is maintained under conditions of mechanical strain is incompletely understood. NOMO is an ER-resident transmembrane protein that contributes to ER morphology and is highly expressed in striated muscle. In this study, we identify a critical interface between distal Ig domains that enables NOMO to maintain ER morphology and buffer mechanical forces. By incorporating two independent tension sensors in the luminal domain of NOMO, we demonstrate that NOMO assemblies experience forces in the single piconewton (pN) range, with a significant contribution from the identified interface. These newly defined features are important, if not indispensable, for myogenesis, as interface mutations affecting mechanosensing fail to restore the essential role of NOMO during myogenesis in a C2C12 differentiation model. Moreover, NOMO depletion impairs nematode motility, underscoring a broader functional importance in muscle physiology.

cell biology↗

bioRxiv2024

Vesiculation pathways in clathrin-mediated endocytosis

During clathrin-mediated endocytosis, a patch of flat plasma membrane is internalized to form a vesicle. In mammalian cells, how the clathrin coat deforms the membrane into a vesicle remains unclear and two main hypotheses have been debated. The "constant area" hypothesis assumes that clathrin molecules initially form a flat lattice on the membrane and deform the membrane by changing its intrinsic curvature while keeping the coating area constant. The alternative "constant curvature" hypothesis assumes that the intrinsic curvature of the clathrin lattice remains constant during the formation of a vesicle while the surface area it covers increases. Previous experimental studies were unable to unambiguously determine which hypothesis is correct. In this paper, we show that these two hypotheses are only two extreme cases of a continuum spectrum if we account for the free energies associated with clathrin assembly and curvature generation. By tracing the negative gradient of the free energy, we define vesiculation pathways in the phase space of the coating area and the intrinsic curvature of clathrin coat. Our results show that, overall, the differences in measurable membrane morphology between the different models are not as big as expected, and the main differences are most salient at the early stage of endocytosis. Furthermore, the best fitting pathway to experimental data is not compatible with the constant-curvature model and resembles a constant-area-like pathway where the coating area initially expands with minor changes in the intrinsic curvature, later followed by a dramatic increase in the intrinsic curvature and minor change in the coating area. Our results also suggest that experimental measurement of the tip radius and the projected area of the clathrin coat will be the key to distinguish between models.

cell biology↗

bioRxiv2024

CRISPR-Cas9 editing efficiency in fission yeast is not limited by homology search and is improved by combining gap-repair with fluoride selection

Protocols for CRISPR-Cas9 editing have been implemented in most model organisms, including fission yeast, for which some improvements have also been later described. Here, we report an improvement to the CRISPR-Cas9 protocol in fission yeast, as we combine a cloning free gap-repair method with our previously described fluoride selection marker, which speeds up genome editing. We also report a wide variability of editing efficiencies at different loci along the genome, and we demonstrate that this variability cannot be explained by the location of the edited sequences in the genome. Lastly, our attempt at improving editing efficiency by targeting the donor DNA to the cut site using a HaloTag strategy to link the donor DNA to two proteins of the homologous recombination repair machinery (Rad51 or Rad52) fell short, which shows that editing efficiency in fission yeast is likely not limited by homology search.

cell biology↗

bioRxiv2023

A mechanical circuit in End4p coordinates force transmission during clathrin-mediated endocytosis

Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe. The onset and termination of force transmission is tightly regulated temporally during different stages of CME, and spatially over the surface of the invaginated membrane. How force transmission is regulated and coordinated at the molecular scale is unclear. An adaptor protein in CME, End4p, directly transmits force by binding to both the membrane (through ANTH domain) and F-actin (through THATCH domain). We show that 8pN is required for stable binding between THATCH and F-actin. We also report the discovery and characterization of a new domain on End4p, which we named Rend (R domain in End4p), that resembles R12 of talin. Membrane localization of Rend primes the binding of THATCH to F-actin, and force-induced unfolding of Rend at 15pN terminates the transmission of force during CME. We show that the mechanical properties (mechanical stability, unfolding length, hysteresis) of Rend and THATCH are tuned to form an auto-regulated circuit for the initiation, transmission and termination of force between the actin cytoskeleton and membrane. Shorting the circuit leads to permanent End4p association with the membrane or with F-actin, or failure to enter the force transmission cycle. Mathematical modeling of force transmission through Rend-THATCH connection shows that input force from F-actin is buffered to a narrow range towards the membrane. The mechanical circuit by Rend and THATCH may be conserved and coopted evolutionarily in cell adhesion complexes.

cell biology↗

bioRxiv2022

Fast actin disassembly and fimbrin mechanosensitivity support rapid turnover during clathrin-mediated endocytosis

The actin cytoskeleton is central to force production in numerous cellular processes in eukaryotic cells. During clathrin-mediated endocytosis (CME), a dynamic actin meshwork is required to deform the membrane against high membrane tension or turgor pressure. Previous experimental work from our lab showed that several endocytic proteins, including actin and actin-interacting proteins, turn over several times during the formation of a vesicle during CME in yeast, and their dwell time distributions were reminiscent of Gamma distributions with a peak around 1 s (Lacy et al., 2019). However, the distribution for the filament crosslinking protein fimbrin contains a second peak around 0.5 s. To better understand the nature of these dwell time distributions, we developed a stochastic model for the dynamics of actin and its binding partners. Our model demonstrates that very fast actin filament disassembly is necessary to reproduce experimental dwell time distributions. Our model also predicts that actin-binding proteins bind rapidly to nascent filaments and filaments are fully decorated. Last, our model predicts that fimbrin detachment from actin endocytic structures is mechanosensitive to explain the extra peak observed in the dwell time distribution.

cell biology↗

bioRxiv2021

A model of actin-driven endocytosis explains differences of endocytic motility in budding and fission yeast

A comparative study (Sun et al., eLife, 2019) showed that the abundance of proteins at sites of endocytosis in fission and budding yeast is more similar in the two species than previously thought, yet membrane invaginations in fission yeast elongate two-fold faster and are nearly twice as long as in budding yeast. Here we use a three-dimensional model of a motile endocytic invagination (Nickaeen et al., MBoC, 2019) to investigate factors affecting elongation of the invaginations. We found that differences in turgor pressure in the two yeast species can largely explain the paradoxical differences observed experimentally in endocytic motility.

cell biology↗

bioRxiv2021

A gradient of force in the endocytic coat is revealed by new coiled-coil force sensors

Forces are central to countless cellular processes, yet in vivo force measurement at the molecular scale remains difficult if not impossible. During clathrin-mediated endocytosis, forces produced by the actin cytoskeleton are transmitted to the plasma membrane by a multi-protein coat for membrane deformation. However, the magnitudes of these forces remain unknown. Here, we present new in vivo force sensors that induces protein condensation under force. We measured the forces on the fission yeast HIP1R homologue End4p, a protein that links the membrane to the actin cytoskeleton. End4p is under [~]19 pN force near the actin cytoskeleton, [~] 11 pN near the clathrin lattice, and [~]9 pN near the plasma membrane. Our results demonstrate that forces are collected and redistributed across the endocytic machinery. One-Sentence SummaryNew in vivo coiled-coil force sensors reveal force redistribution during endocytosis.

cell biology↗

bioRxiv2020

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in mammalian and bacterial cells, tagged with fluorescent proteins or organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

biophysics↗

bioRxiv2019

Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

Actin is required for clathrin-mediated endocytosis (CME) in yeast. Experimental observations indicate that this actin assembly generates force to deform the membrane and overcome the cells high turgor pressure, but the precise molecular details remain unresolved. Based on previous models, we predicted that actin at endocytic sites continually polymerize and disassemble, turning over multiple times during an endocytic event. Here we applied single-molecule speckle tracking in live fission yeast to directly measure this predicted turnover within the CME assembly for the first time. In contrast with the overall ~20-sec lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 sec, and high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins motions. These results indicate that rapid and continuous turnover is a key feature of the endocytic machinery and suggest revising quantitative models of force production.

cell biology↗

bioRxiv2019

Crosslinking actin networks produces compressive force

Actin has been shown to be essential for clathrin-mediated endocytosis in yeast. However, actin polymerization alone is likely insufficient to produce enough force to deform the membrane against the huge turgor pressure of yeast cells. In this paper, we used Brownian dynamics simulations to demonstrate that crosslinking of a meshwork of non-polymerizing actin filaments is able to produce compressive forces. We show that the force can be up to thousands of piconewtons if the crosslinker has a high stiffness. The force decays over time as a result of crosslinker turnover, and is a result of converting chemical binding energy into elastic energy.

biophysics↗

bioRxiv2019

Mechanics of membrane internalization against osmotic pressure: boundary conditions make a difference

During clathrin-mediated endocytosis, a patch of flat plasma membrane is deformed into a vesicle. In walled cells, such as plants and fungi, the turgor pressure is high and pushes the membrane against the cell wall, thus hindering membrane internalization. In this paper, we study how a patch of membrane is deformed against turgor pressure by force and by curvature-generating proteins. We show that a large amount of force is needed to merely start deforming the membrane and an even larger force is needed to pull a membrane tube. The magnitude of these forces strongly depends on how the base of the membrane is constrained and how the membrane is coated with curvature-generating proteins. In particular, these forces can be reduced by partially but not fully coating the membrane patch with curvature-generating proteins. Our theoretical results show excellent agreement with experimental data. SIGNIFICANCEYeast cells have been widely used as a model system to study clathrin-mediated endocytosis. The mechanics of membrane during endocytosis has been extensively studied mostly in low turgor pressure condition, which is relevant for mammalian cells but not for yeast cells. It has been suggested that as a result of high turgor pressure in yeast cells, a large amount of force is needed to drive the progress of the membrane invagination. In this paper, we investigated biologically relevant mechanisms to reduce the force requirement. We highlight the role of boundary conditions at the membrane base, which is a factor that has been largely ignored in previous studies. We also investigate the role of curvature-generating proteins and show that a large protein coat does not necessarily reduce the force barrier for endocytosis.

biophysics↗

bioRxiv2019

Simulation of the mechanics of actin assembly during endocytosis in yeast

We formulated a spatially resolved model to estimate forces exerted by a polymerizing actin meshwork on an invagination of the plasma membrane during endocytosis in yeast cells. The model is a continuous approximation tightly constrained by experimental data. Simulations of the model produce forces that can overcome resistance of turgor pressure in yeast cells. Strong forces emerge due to the high density of polymerized actin in the vicinity of the invagination and because of entanglement of the meshwork due to its dendritic structure and crosslinking. The model predicts forces orthogonal to the invagination that would result in a flask shape that diminishes the net force due to turgor pressure. Simulations of the model with either two rings of nucleation promoting factors as in fission yeast or a single ring of nucleation promoting factors as in budding yeast produce enough force to elongate the invagination against the turgor pressure.

cell biology↗

bioRxiv2018

Conditions for optimal mechanical power generation by a bundle of growing actin filaments

Bundles of actin filaments are central to a large variety of cellular structures, such as filopodia, stress fibers, cytokinetic rings or focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles rigidity and shape. However, it remains unknown how bundle length and thickness are controlled by external physical factors, and how the attachment of the bundle to a load affects its ability to transmit forces. In this paper, we present a biophysical model for dynamic bundles of actin filaments that takes into account individual filaments, their interaction with each other and with an external load. In combination with in vitro motility assays of beads coated with formins, our model allowed us to characterize conditions for bead movement and bundle buckling. From the deformation profiles, we determined key biophysical properties of tethered actin bundles, such as their rigidity and filament density. Our model also demonstrated that filaments undulate under lateral constraints applied by external forces or by neighboring filaments of the bundle. Last, our model allowed us to identify optimum conditions in filament density and barbed end tethering to the load for a maximal yield of mechanical power by a dynamic actin bundle.

biophysics↗