Biology subjects

Bassereau, P.

Publications and source records attributed to Bassereau, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Subdomains of Endophilin-NBAR Can Synergistically Drive Membrane Remodeling and Facilitate Controlled Membrane Scission

The NBAR-domain containing protein endophilin, as a major player in many endocytic pathways, has offered considerable insight into BAR-domain driven membrane remodeling. However, understanding the interaction of the different subdomains of endophilin and their abilities to sense and generate negative Gaussian curvature are yet unanswered questions, with significant implications for the mechanisms and regulation of unconventional endocytic pathways. Using coarse-grained molecular dynamics simulation, we demonstrate the synergistic remodeling capabilities of the NBAR remodeling unit, as well as its ability to sort to and generate membrane regions with negative Gaussian curvature. We find that the assembly of NBAR scaffolds at regions of negative Gaussian curvature facilitate membrane hemifission in dynamic bud formation. These insights provide an additional role for endophilin scaffolds in endocytosis, as well as emphasizing the importance of developing new ways to study negative Gaussian curvature. STATEMENT OF SIGNIFICANCEThis work provides deeper insight into the composite membrane remodeling abilities of NBAR domains in peripheral membrane proteins and their sorting to negative Gaussian curvature. Theis work also explicitly models at the molecular level the {Omega}-shaped membrane geometry, connecting endophilin mechanics to its physiological function in endocytosis.

biophysics↗

bioRxiv2025

Palmitoylated caveolin-1 enables endosome sorting of complex sphingolipids

Complex sphingolipids form in the Golgi apparatus and require transport by vesicular carriers to reach the plasma membrane (PM) where they assemble with cholesterol to affect membrane function. The caveolin proteins have been implicated in sphingolipid trafficking but by mechanisms unknown. Here, we found that cells lacking caveolin-1 (Cav1) distributed the complex sphingolipids to the lysosome rather than to the PM. This was not seen in Cavin-1 KO cells, implicating a function for Cav1 independent of caveolae. The defect in trafficking localized to the sorting endosome where the complex sphingolipids failed to enter recycling tubules serving the PM. Sphingolipid trafficking was rescued by over-expression of Cav1, but not by a Cav1 mutant that lacked the S-palmitoylation sites. Thus, noncaveolar and palmitoylated Cav1 appears to act as a chaperone, or selectivity filter, enabling entry of the complex sphingolipids into endocytic recycling tubules to shape the composition of the PM.

cell biology↗

bioRxiv2025

The PXBAR-domain protein SNX9 selectively sequesters PI(3,4)P2 lipid and protects it from hydrolysis at the plasma membrane

Plasma membrane remodeling processes are tightly regulated by the spatiotemporal distribution and dynamic conversion of phosphoinositidyl lipids (PIPs). This regulation is controlled by the recruitment of proteins such as sorting nexin 9 (SNX9), a key mediator of late-stage endocytosis and macropinocytosis. Using live cell imaging, in vitro reconstitution assays, and molecular dynamics simulations, we investigated how SNX9 distinguishes between PI(3,4)P2 and PI(4,5)P2, and the physiological relevance of this selectivity. Our results revealed that during macropinocytic membrane ruffling, SNX9 is recruited in a spatiotemporally coordinated manner with PI(3,4)P2, but not with PI(4,5)P2. While SNX9 induces comparably weak mechanical remodeling on model membranes containing either PIP2 species, it exhibits a clear selective binding to PI(3,4)P2, mediated by a non-canonical interface. Through mutational analysis of key residues involved in this sequestration, we further demonstrated that SNX9 protects PI(3,4)P2 from hydrolysis. Together, these results reveal a previously unrecognized mechanism of SNX9-PIP2 lipid interaction that underscores SNX9s pivotal role in coordinating membrane remodeling processes. TeaserCurvature sensing BAR protein SNX9 selectively sequesters PI(3,4)P2 lipids, acting as a checkpoint in cell membrane remodeling.

biophysics↗

bioRxiv2024

Membrane curvature association of amphipathic helix 8 drives constitutive GPCR endocytosis

Cellular signaling relies on the activity of transmembrane receptors and their presentation on the cellular surface. Their continuous insertion in the plasma membrane is balanced by constitutive and activity dependent internalization, which is orchestrated by adaptor proteins recognizing semi-specific motifs within the receptors intracellular regions. Here we describe a complementary and evolutionary conserved and refined trafficking mechanism for G-protein coupled receptors (GPCR). This mechanism relies on the insertion of their amphipathic helix 8 into the inner leaflet of lipid membranes, orthogonal to the transmembrane helices. These amphipathic helices dictate subcellular localization of the receptors and autonomously drive their endocytosis by cooperative assembly and association with areas of high membrane curvature. The strength of helix 8 membrane insertion propensity quantitatively predicts the rate of constitutive internalization of GPCRs. This discovery advances our understanding of membrane protein trafficking and highlights a new principle of receptor-lipid interactions that may have broader implications for cellular signaling and therapeutic targeting. One-Sentence SummaryReceptor proteins navigate cellular membranes by interacting with their curvature using an evolutionary conserved mechanism that relies on amphipathic helices and complements direct coupling to the endocytic protein machinery.

cell biology↗

bioRxiv2024

STRIPAK controls cell-cell communication by promoting cytoneme biogenesis through the membrane-sculpting function of Slik.

Cytonemes are signaling filopodia that facilitate long-range cell-cell communication by forming synapses between cells. Initially discovered in Drosophila for transporting morphogens during embryogenesis, they have since been identified in mammalian cells and recently implicated in carcinogenesis. Yet, despite their importance, the mechanisms controlling cytoneme biogenesis remain elusive. Here, we demonstrate that the Ser/Thr kinase Slik drives remote cell proliferation by promoting cytoneme formation. We discovered that this function depends on the coiled-coil domain of Slik (SlikCCD), which directly sculpts membranes into tubules. Importantly, Slik plays paradoxical roles in cytoneme biogenesis. While its membrane-sculpting activity promotes cytoneme formation, it is counteracted by its kinase activity, which enhances actin association with the plasma membrane via Moesin phosphorylation. In vivo, SlikCCD enhances formation of cytonemes in one epithelial layer of the wing disc to promote cell proliferation in an adjacent layer. Finally, we found that this function relies on the STRIPAK complex, which controls cytoneme formation and governs proliferation at a distance by regulating Slik association with the plasma membrane. Our study unveils the first family of kinases that directly sculpts membranes, a function crucial for cytoneme-mediated control of cell proliferation.

cell biology↗

bioRxiv2024

Mechanosensitivity of an ABC Membrane Transporter Revealed by Single Molecule FRET and Activity Measurement

While mechanosensitive ion channels gating has been well documented, the effect of membrane mechanics, in particular membrane curvature, on the function of transporters remains elusive. Since conical shape transmembrane proteins locally deform membranes, conversely membrane bending could impact their conformations and their function. We tested this hypothesis using BmrA, a bacterial ABC exporter that exhibits large conformational changes upon ATP hydrolysis, switching between open and closed states with opposite V-shapes. After reconstitution in liposomes of different curvatures, and at two different temperatures, we showed that BmrA ATPase activity decreases by 2.9-fold when their diameter decreases from 125 to 29 nm. Moreover, using single-molecule FRET, we observed that the fraction of closed conformations is reduced in highly curved vesicles when adding ATP or non-hydrolysable AMP-PNP. Our results are well explained by a theoretical 2-states model including the effect of membrane mechanics on protein shape transition. Our work reveals that the functional cycle of conical transporters is curvature sensitive, to an extent depending on protein geometry. Significance StatementBiological membranes actively regulate protein function. While some ion channels are known to sense membrane tension, whether transporters also respond to mechanical cues was unknown. We show that the conical-shaped bacterial exporter BmrA is sensitive to membrane curvature, with both its activity and conformation landscape determined by the surrounding lipid bilayer. This reveals a previously unrecognized mechanism of mechanosensitivity, suggesting that membrane geometry and transporter shape are intrinsically coupled, providing a general principle of regulation across biological systems.

biophysics↗

bioRxiv2022

Caveolae govern plasma membrane mechanics to protect cells against EDIN B-induced transcellular tunnel formation and lethality from S. aureus septicaemia

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point towards membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction of cell height, due to the increase of cell spreading and decrease of cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs leading to their dramatic 5.4-fold larger widening. Consistent with the increase of TEM density and width in siCAV1 cells, we record a higher lethality in caveolin-1-deficient mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity point toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

biophysics↗

bioRxiv2022

Cell adhesion and spreading on fluid membranes through microtubules-dependent mechanotransduction

During cell adhesion, integrins form clusters that transmit mechanical forces to the substrate (mechanotransduction) and regulate biochemical signaling depending on substrate stiffness. Studies on mechanotransduction significantly advanced our understanding of cell adhesion and were mostly performed on rigid substrates. In contrast to rigid substrates, integrins ligands on fluid supported lipid bilayers (SLBs) are mobile and adhesive complexes cannot serve as anchoring points promoting cell spreading. Here, we demonstrate that cells spread on SLBs coated with Invasin, a high-affinity integrin ligand. We show that in contrast to SLBs functionalized with RGD peptides, integrin clusters grow in size and complexity on Invasin-SLBs to a similar extent as on glass. While actomyosin contraction dominates adhesion maturation on stiff substrates, we find that integrin mechanotransduction and cell spreading on fluid SLBs rely on dynein pulling forces along microtubules perpendicular to membranes and microtubules pushing on adhesive complexes, respectively. These forces that may also occur on non-deformable surfaces are revealed in fluid substrate set ups. Our findings, supported by a theoretical model, demonstrate a new mechanical role for microtubules in integrin clustering.

cell biology↗

bioRxiv2022

Arp2/3 inhibition switches Eps8's network associations to favour longer actin filament formation necessary for tunneling nanotubes

Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we demonstrate that TNTs forming through the outward extension of actin (not through cellular dislodgement) achieve distances greater than the mean length of filopodia, and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, limiting TNT occurrence. Proteomic analysis using Epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form TNTs, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour TNT formation. Thus, this work reinforces a general principle for actin network control for cellular protrusions where simple shifts in the balance between processes that inhibit actin growth versus those that promote growth dictate protrusion formation and the ultimate length scales protrusions achieve.

cell biology↗

bioRxiv2022

Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo electron microscopy structures of membrane-coated CHMP2A-CHMP3 filaments of two different diameters at 3.3 and 3.6 [A] resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence towards the tube interior. Inter-filament interactions are electrostatic, which facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high speed atomic force microscopy imaging corroborate that CHMP2A-CHMP3 polymers and VPS4 can constrict and cleave narrow membrane tubes, thus acting as a minimal membrane fission machinery.

cell biology↗

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

Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodia initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro and in silico to unravel the mechanism of filopodia initiation driven by the membrane curvature sensor IRSp53. We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodia initiation.

biophysics↗

bioRxiv2022

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ( reverse-topology), but they can also be directed towards the cytosol ( normal-topology). Of the ESCRT complexes 0-III, ESCRT-III is most directly implicated in membrane severing. Various subunits of ESCRT-III recruit the AAA+ ATPase VPS4, which is essential for ESCRT disassembly and reverse topology membrane scission. The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing, perhaps in conjunction with a AAA+ ATPase. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal topology membrane scission. In order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin, we sought to reconstitute the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles (GUVs) using an optical trap. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1-scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. Spastin colocalized with CHMP1B enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 but did not lead to scission. These data show that the CHMP1B and IST1 tubular coat contributes to membrane scission. Constriction alone is insufficient for scission. However, the dynamical extension of the coated tube does lead to scission. Finally, we find that in the normal topology setting analyzed here, scission is independent of VPS4 and spastin. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism.

biophysics↗

bioRxiv2021

PI4P and BLOC-1 remodel endosomal membranes into tubules

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers play major roles in the flux of membrane lipids and proteins to acceptor organelles. However, how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. By exploiting imaging approaches at different scales on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability and functions of recycling endosomal tubules. Endosomal PI4P production by type II PI4-kinases is needed to form nascent curved tubules through binding of BLOC-1 that stabilize and elongate them. Membrane remodeling by the PI4P/ BLOC-1 module functions not only in the recycling of endosomal cargoes, but also in the lifecycles of intracellular pathogens such as Chlamydia bacteria and influenza virus. This study demonstrates how a phospholipid and a protein complex coordinate as a minimal machinery to remodel cellular membranes into functional tubes.

cell biology↗

bioRxiv2021

Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53.

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. Partial gene editing of IRSp53 induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein localizes preferentially to IRSp53 I-BAR domain induced curved membranes on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

biophysics↗

bioRxiv2020

Influence of membrane-cortex linkers on the extrusion of membrane tubes

The cell membrane is an inhomogeneous system composed of phospholipids, sterols and proteins that can be directly attached to underlying cytoskeleton. The linkers between the membrane and the cytoskeleton are believed to have a profound effect on the mechanical properties of the cell membrane and its ability to reshape. Here we investigate the role of membrane-cortex linkers on the extrusion of membrane tubes using computer simulations and experiments. In simulations we find that the force for tube extrusion has a non-linear dependence on the density of membrane-cortex attachments: at a wide range of low and intermediate densities of linkers the force is not significantly influenced by the presence of membrane linking proteins and resembles that of the bare membrane. For large concentrations of linkers however the force substantially increases compared to the bare membrane. In both cases the linkers provided membrane tubes with increased stability against coalescence. We then pulled tubes from HEK cells using optical-tweezers for varying expression levels of the membrane-cortex attachment protein Ezrin. In line with simulations, we observed that overexpression of Ezrin led to an increased extrusion force, while Ezrin depletion had negligible effect on the force. Our results shed light on the importance of local effects in membrane reshaping at the nanoscopic scales.

biophysics↗

bioRxiv2020

Membrane-mediated Forces Can Stabilize Tubular Assemblies of I-BAR Proteins

Collective action by Inverse-BAR (I-BAR) domains drive micron-scale membrane remodeling. The macroscopic curvature sensing and generation behavior of I-BAR domains is well characterized, and computational models have suggested various mechanisms on simplified membrane systems, but there remain missing connections between the complex environment of the cell and the models proposed thus far. Here, we show a connection between the role of protein curvature and lipid clustering in the stabilization of large membrane deformations. We find lipid clustering provides a directional membrane-mediated interaction between membrane-bound I-BAR domains. Lipid clusters stabilize I-BAR domain aggregates that would not arise through membrane fluctuation-based or curvature-based interactions. Inside of membrane protrusions, lipid cluster-mediated interaction draws long side-by-side aggregates together resulting in more cylindrical protrusions as opposed to bulbous, irregularly shaped protrusions. Statement of SignificanceMembrane remodeling occurs throughout the cell and is crucial to proper cellular function. In the cellular environment, I-BAR proteins are responsible for sensing membrane curvature and initiating the formation of protrusions outward from the cell. Additionally, there is a large body of evidence that I-BAR domains are sufficient to reshape the membrane on scales much larger than any single domain. The mechanism by which I-BAR domains can remodel the membrane is uncertain. However, experiments show that membrane composition and most notably negatively-charge lipids like PIP2 play a role in the onset of tubulation. Using coarse-grained models, we show that I-BAR domains can cluster negatively charge lipids and clustered PIP2-like membrane structures facilitate a directional membrane-mediated interaction between I-BAR domains.

biophysics↗

bioRxiv2020

Myosin 1b Flattens and Prunes Branched Actin Filaments.

Motile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Using in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show in this report that this molecular motor flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and in the dynamics of actin networks in different cellular regions. Short summaryUsing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show that myosin flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches

biophysics↗