Biology subjects

Roux, A.

Publications and source records attributed to Roux, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Friction patterns guide actin network contraction

The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile acto-myosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we micropatterned actin nucleation promoting factors on glass or on a lipid bilayer. We adapted their concentrations on each surface to induce the assembly of actin networks of similar densities, and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction both the active and resistive forces are essential to direct the actin network deformation. Significance statementCell shape changes are controlled by complex sets of mechanical forces of various origins. Numerous studies have been dedicated to the role of active forces, originating from molecular motors and filament polymerization, but much less is known about the guiding role of resistive forces. Here we show that a non-uniform distribution of friction forces between a contracting acto-myosin network and its underlying substrate can direct its deformation as it contracts. Our results suggest that the contribution of resistive forces, such as anchoring forces but also less specific viscous forces along cell surface, can be as significant as those of active forces in driving network deformation and should be considered in mechanical models describing the regulation of cell shape and movements.

biophysics↗

bioRxiv2022

Modelling Membrane Reshaping by Staged Polymerization of ESCRT-III Filaments

ESCRT-III filaments are composite cytoskeletal polymers that can constrict and cut cell membranes from the inside of the membrane neck. Membrane-bound ESCRT-III filaments undergo a series of dramatic composition and geometry changes in the presence of an ATP-consuming Vps4 enzyme, which causes stepwise changes in the membrane morphology. We set out to understand the physical mechanisms involved in translating the changes in ESCRT-III polymer composition into membrane deformation. We have built a coarse-grained model in which ESCRT-III polymers of different geometries and mechanical properties are allowed to copolymerise and bind to a deformable membrane. By modelling ATP-driven stepwise depolymerisation of specific polymers, we identify mechanical regimes in which changes in filament composition trigger the associated membrane transition from a flat to a buckled state, and then to a tubule state that eventually undergoes scission to release a small cargo-loaded vesicle. We then characterise how the location and kinetics of polymer loss affects the extent of membrane deformation and the efficiency of membrane neck scission. Our results identify the near-minimal mechanical conditions for the operation of shape-shifting composite polymers that sever membrane necks. Author summaryESCRT-III proteins have the unique ability to cut membrane necks from within, which is needed for a vast number of cell remodelling events including the release of cargo-containing vesicles. ESCRT-III proteins exist in different forms, which can assemble into spiral and helical homopolymers of different curvatures, and they have been suggested to polymerize and depolymerize with each other in a staged manner to deform and cut membranes. We developed a computer model to explore the physical mechanisms behind vesicle budding driven by the staged assembly and disassembly of multiple elastic filaments. We identified rules that determine the outcomes of membrane remodelling, which depend on the relative physical features of the distinct filaments, the dynamics of their disassembly, and on the presence of cargo; thereby providing experimentally testable predictions. Our study provides new physical insights into the ESCRT-III-mediated vesicle budding process, at a single subunit level, and identifies the general design principles of nanomachines built from shapeshifting copolymers, which might also be realized in synthetic systems.

biophysics↗

bioRxiv2022

Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes

Measuring forces within living cells remains a technical challenge. We developed hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing and can report on membrane tension. Here, we describe technical optimization of the probe imaging, and diverse characterizations in various biological and in vitro systems. We provide a guideline to measure biophysical parameters of cellular membranes by FLIM microscopy with Flipper probes, providing evidences that flippers can report long range forces in cells, tissues and organsi.

biophysics↗

bioRxiv2022

Retromer oligomerization drives SNX-BAR coat assembly and membrane constriction

The retromer coat mediates protein exit from endosomes and impacts many signaling pathways, lysosomal biogenesis, and diseases such as Parkinsons, Alzheimers and COVID-19. Retromer complexes (CSC in yeast) form coats by interconnecting sorting nexins (SNX). The dynamics of this process is poorly explored. Here, we analyze the oligomerization of CSC/SNX-BAR retromer coats on oriented synthetic lipid tubules. SNX-BARs and CSC assemble a static tubular coat that does not exchange subunits. Coat formation proceeds bidirectionally, adding new subunits at both ends of the coat. High concentrations of SNX-BARs alone suffice to constrict membrane tubes to an invariant radius of 19 nm. At lower concentrations, CSC-complexes must drive constriction, which requires their oligomerization. CSCs populate the SNX-BAR layer at densities that increase with the starting radius of the membrane tube. We hence propose that retromer-mediated crosslinking of SNX-BARs at variable densities tunes the coat according to the energy required to deform the membrane. This model is supported by the effects of mutations interfering with retromer oligomerization, which impair retromer function in yeast and human cells.

cell biology↗

bioRxiv2022

Mechanochemical rules for membrane-reshaping composite filaments

The sequential exchange of filament composition to increase filament curvature was proposed as a mechanism for how ESCRT-III polymers deform and cut membranes. The relationship between the filament composition and its mechanical effect is lacking. We develop a kinetic model for the assembly of composite filaments that includes protein-membrane adhesion, filament mechanics and membrane mechanics. We identify the physical conditions for such a membrane remodelling and show this mechanism is efficient because sequential polymer assembly lowers the energetic barrier for membrane deformation.

biophysics↗

bioRxiv2022

Vps60 initiates formation of alternative membrane-bound ESCRT-III filaments

Endosomal sorting complex required for transport-III (ESCRT-III)-driven membrane remodeling participates in many crucial cellular functions, from cell division to endosome maturation, and occurs on essentially all cellular organelles. In eukaryotes, ESCRT-III displays a remarkable molecular diversity in its subunits which may have been acquired through evolution to perform novel cellular functions. Here, we describe and characterize a novel ESCRT-III polymer initiated by the subunit Vps60. Membrane-bound Vps60 polymers recruit ESCRT-III subunits Vps2, Vps24, Did2 and Ist1, and undergo polymer turnover powered by the ATPase Vps4. Snf7- and Vps60 filaments can coexist on membranes without interacting. Their nucleation, polymerization and recruitment of downstream subunits remains unaffected by the presence of the respective other polymer. Taken together, our results suggest Vps60 and Snf7 form distinct ESCRT-III polymers, which overall, supports the notion of evolutionary diversification of ESCRT-III assemblies to perform specific cellular functions.

cell biology↗

bioRxiv2022

Neuro-toxicogenomic mapping of TMT induced neurotoxicity using human minibrain reveals associated adverse outcome events

Genome-wide transcriptomic interrogation of organoids and 3D tissue models are increasingly used for characterizing drug, toxicity responses and neurodevelopmental disorders. We established here a neuro-toxicogenomic assay by utilizing "minibrain", a human in vitro 3D brain model system and a low-cost, highly multiplexable RNA-seq methodology (BRB-seq) for screening the effect of trimethyltin chloride (TMT) induced neurotoxicity. We demonstrate that transcriptomic profiling is insightful to the cellular composition and regional identity of the minibrain. Further, we characterize the transcriptomic changes associated with the dose-time neurotoxic response of minibrain upon exposure to TMT. The distinct gene expression changes and molecular candidates identified with our pipeline provides insight to map the key events involved in the adverse outcome pathway of TMT associated neurotoxicity. We identify processes such as endoplasmic reticulum stress, dysregulation of synaptic genes and downregulation of neuron-morphology associated genes upon exposure to TMT. In response to TMT, we identify activation of an early response homeostatic mechanism in minibrain and an interplay of STAT pathways correlating with the dose severity. In this study, we present a neuro-toxicogenomic assay that demonstrates the power of a low-cost transcriptomic screening to study chemical induced neurotoxicity.

neuroscience↗

bioRxiv2021

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

bioRxiv2021

Pressure and curvature control of contact inhibition in epithelia growing under spherical confinement

Morphogenesis requires spatiotemporal regulation of cell shape and proliferation, both regulated by biochemical and mechanical cues. In epithelia, this regulation is called contact inhibition, but disentangling biochemical from mechanical cues remains challenging. Here, we show that epithelia growing under confinement accumulate pressure that inhibits proliferation above a threshold value, which depends on the {beta}-catenin pathway. Before inhibition of proliferation, cell aspect ratio abruptly increased upon reaching confluency. This shape transition occurred at low, constant pressure and was mainly controlled by cell density and contractility, correlating with YAP/TAZ pathway inhibition. In our system, epithelia spontaneously buckle: we observed that folding transiently reactivates both the YAP/TAZ pathway and cell proliferation. Altogether, our results support that different mechanical cues part of contact inhibition regulate cell proliferation through different mechanosensing pathways. Proliferation is regulated by sustained, tissue-level pressure through the {beta}-catenin pathway, and by local curvature and pressure changes through the YAP/TAZ pathway.

cell biology↗

bioRxiv2021

Partial reprogramming restores youthful gene expression through transient suppression of cell identity

Transient induction of pluripotent reprogramming factors has been reported to reverse some features of aging in mammalian cells and tissues. However, the impact of transient reprogramming on somatic cell identity programs and the necessity of individual pluripotency factors remain unknown. Here, we mapped trajectories of transient reprogramming in young and aged cells from multiple murine cell types using single cell transcriptomics to address these questions. We found that transient reprogramming restored youthful gene expression in adipocytes and mesenchymal stem cells but also temporarily suppressed somatic cell identity programs. We further screened Yamanaka Factor subsets and found that many combinations had an impact on aging gene expression and suppressed somatic identity, but that these effects were not tightly entangled. We also found that a transient reprogramming approach inspired by amphibian regeneration restored youthful gene expression in aged myogenic cells. Our results suggest that transient pluripotent reprogramming poses a neoplastic risk, but that restoration of youthful gene expression can be achieved with alternative strategies.

systems biology↗

bioRxiv2021

Quantitative coupling of cell volume and membrane tension during osmotic shocks

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as a quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase, quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. During these asymmetric recoveries, tension and volume remained coupled. Pharmacological disruption of the cytoskeleton and functional inhibition of ion channels and mTOR all affected tension and volume responses, proving that a passive mechanism is necessary and critical for the cell to adapt fast. The coupling between them was, nonetheless, maintained for a few exceptions suggesting that volume and tension regulations are independent from the regulation of their coupling.

biophysics↗

bioRxiv2020

Transient active osmotic swelling of epithelium upon curvature induction

Generation of tissue curvature is essential to morphogenesis. However, how cells adapt to changing curvature is still unknown because tools to dynamically control curvature in vitro are lacking. Here we developed self-rolling substrates to study how flat epithelial cell monolayers adapt to a rapid, anisotropic change of curvature. We show that the primary response is an active and transient osmotic swelling of cells. This cell volume increase is not observed on inducible wrinkled substrates, where concave and convex regions alternate each other over short distances, identifying swelling as a collective response to changes of curvature with persistent sign over large distances. It is triggered by a drop in membrane tension and actin depolymerization, perceived by cells as a hypertonic shock. Osmotic swelling restores tension while actin reorganizes, probably to comply with curvature. Epithelia are thus unique materials that transiently, actively swell while adapting to large curvature induction.

biophysics↗

bioRxiv2020

Mass Generation, Neuron Labelling and 3D Imaging of Minibrains

Minibrain is a spherical in vitro 3D brain organoid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human brain organoid models, there is a lack of labelling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains. Using the pipeline, we present i. dendrite morphometric properties obtained from 3D neuron morphology reconstructions and ii. distribution and quantification of cell types in 3D across whole mount organoids.

neuroscience↗

bioRxiv2020

Integer topological defects organize stresses driving tissue morphogenesis

Tissues acquire their function and shape via differentiation and morphogenesis. Both processes are driven by coordinating cellular forces and shapes at the tissue scale, but general principles governing this interplay remain to be discovered. Here, we report that self-organization of myoblasts around integer topological defects, namely spirals and asters, triggers localized differentiation and, when differentiation is inhibited, drives the growth of cylindrical multicellular protrusions. Both localized differentiation and growth require specific stress patterns. By analyzing the experimental velocity and orientation profiles through active gel theory, we show that integer topological defects can concentrate compressive stresses, which we measure by using deformable pillars. Altogether, we envision topological defects as mechanical organizational centers that control differentiation and morphogenesis to establish tissue architecture.

biophysics↗

bioRxiv2020

Cingulin unfolds ZO-1 and organizes myosin-2B and gamma-actin to mechanoregulate apical and tight junction membranes

How junctional proteins regulate the mechanics of the plasma membrane and how actin and myosin isoforms are selectively localized at epithelial cell-cell junctions is poorly understood. Here we show by atomic force indentation microscopy, immunofluorescence analysis and FLIM membrane tension imaging that the tight junction (TJ) protein cingulin maintains apical surface stiffness and TJ membrane tortuosity and down-regulates apico-lateral membrane tension in MDCK cells. KO of cingulin in MDCK, mCCD and Eph4 cells results in a decrease in the juxta-membrane accumulation of labeling for cytoplasmic myosin-2B (NM2B), {gamma}-actin, phalloidin and ARHGEF18, but no detectable effect on myosin-2A (NM2A) and {beta}-actin. Loss of paracingulin leads to weaker mechanical phenotypes in MDCK cells, correlating with no detectable effect on the junctional accumulation of myosins and actins. Cingulin and paracingulin form biomolecular condensates, bind to the ZU5 domain of ZO-1, and are recruited as clients into ZO-1 condensates in a ZU5-dependent manner. Cingulin binding to ZO-1 promotes the unfolding of ZO-1, as determined by interaction with DbpA in cells lacking ZO-2 and in vitro. Cingulin promotes the accumulation of a pool of ZO-1 at the TJ and is required in a ZU5-dependent manner for the recruitment of phalloidin-labelled actin filaments into ZO-1 condensates, suggesting that ZU5-cingulin interaction promotes ZO-1 interaction with actin filaments. Our results indicate that cingulin tethers the juxta-membrane and apical branched {gamma}-actin-NM2B network to TJ to modulate ZO-1 conformation and the TJ assembly of a pool of ZO-1 and fine-tune the distribution of forces to apical and TJ membranes.

cell biology↗

bioRxiv2020

Tracking crystallophore nucleating properties: setting-up a database for statistical analysis.

In this article, the principle of a database aimed at facilitating the understanding of the unique protein nucleating properties of the Crystallophore is presented. A first analysis allows us to compare the efficiency of Tb-Xo4 with the new Lu-Xo4 variant, featuring improved phasing properties. Then, the concept of subset-of-interest is introduced to reveal potential antagonistic/synergistic effects between Tb-Xo4 and physico-chemical parameters of the crystallisation kits such as pH. The overall approach may be of interest for any studies working on solutions dedicated to improve the nucleating step in protein crystallization. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/057596v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@351291org.highwire.dtl.DTLVardef@5620fcorg.highwire.dtl.DTLVardef@8fadb2org.highwire.dtl.DTLVardef@d4c208_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisA database and associated representation tools are highlighted to understand nucleating properties of the crystallophore.

biochemistry↗

bioRxiv2019

Conserved function of ether lipids and sphingolipids in the early secretory pathway

Sphingolipids have been shown to play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, where depletion of one of these lipid types resulted in increases in the other, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as GPI anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

biochemistry↗

bioRxiv2019

Vps4 triggers sequential subunit exchange in ESCRT-III polymers that drives membrane constriction and fission

ESCRT-III is a ubiquitous complex which catalyzes membrane fission from within membrane necks via an as yet unknown mechanism. Here, we reconstituted in vitro the ESCRT-III complex onto membranes. We show that based on variable affinities between ESCRT-III proteins and the ATPase Vps4, subunits are recruited to the membrane in a Vps4-driven sequence that starts with Snf7 and ends with Did2 and Ist1 which, together, form a fission-active subcomplex. Sequential recruitment of ESCRT-III subunits is coupled to membrane remodeling. Binding of Did2 promoted the formation of membrane protrusions which later constricted and underwent fission mediated by the recruitment of Ist1. Overall, our results provide a mechanism to explain how a sequence of ESCRT-III subunits drives membrane deformation and fission.

biochemistry↗