Biology subjects

Ladoux, B.

Publications and source records attributed to Ladoux, B..

33 records · Page 2Linked to original sources

bioRxiv2021

Mechanics defines the spatial pattern of compensatory proliferation

The number of cells in tissues is tightly controlled by cell division and cell death, and misregulation of cell numbers could lead to pathological conditions such as cancer. To maintain cell numbers in a tissue, a cell elimination process named programmed cell death or apoptosis, stimulates the proliferation of neighboring cells. This mechanism is called apoptosis-induced compensatory proliferation, which was originally reported more than 40 years ago. While only a limited number of the neigboring cells need to divide to compensate for apoptotic cell loss, the mechanisms that select cells for undergoing division remain an open question. Here we found that the spatial inhomogeneity in mechanotransduction through a growth-promoting transcription co-activator Yes-associated protein (YAP) in the neighboring tissue, accounts for the inhomogeneity of compensatory proliferation. Such inhomogeneous mechanotransduction arises from the combination of the non-uniform distribution of nuclear size, which is inherent in tissues, and the non-uniform pattern of mechanical force applied to the neighboring cells upon apoptosis. Our findings from a mechanical perspective complement the current biochemical understanding of compensatory growth and provide additional insights into cellular functions of how tissue precisely maintains its homeostasis.

cell biology↗

bioRxiv2021

Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip For Modelization of Muscular Dystrophies

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for muscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which is problematic in the context of limited patient sample. Here we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments. Spontaneous contractions in myotubes derived from primary human myoblasts were observed. Analysis of nuclear morphology confirmed a similar organization between obtained myotubes and in vivo myofibers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modelled with successful development of mutant 3D myotubes displaying contractile dysfunction. This technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should allow to substantially improve drug screening capability.

bioengineering↗

bioRxiv2021

Cell motility driven by long-lived spatial memory

Many living cells actively migrate in their environment to perform key biological functions - from unicellular organisms looking for food to single cells such as fibroblasts, leukocytes or cancer cells that can shape, patrol or invade tissues. Cell migration results from complex intracellular processes that enable cell self-propulsion 1,2, and has been shown to also integrate various chemical or physical extracellular signals 3,4,5. While it is established that cells can modify their environment by depositing biochemical signals or mechanically remodeling the extracellular matrix, the impact of such self-induced environmental perturbations on cell trajectories at various scales remains unexplored. Here, we show that cells remember their path: by confining cells on 1D and 2D micropatterned surfaces, we demonstrate that motile cells leave long-lived physicochemical footprints along their way, which determine their future path. On this basis, we argue that cell trajectories belong to the general class of self-interacting random walks, and show that self-interactions can rule large scale exploration by inducing long-lived ageing, subdiffusion and anomalous first-passage statistics. Altogether, our joint experimental and theoretical approach points to a generic coupling between motile cells and their environment, which endows cells with a spatial memory of their path and can dramatically change their space exploration.

biophysics↗

bioRxiv2020

Nature of active forces in tissues: how contractile cells can form extensile monolayers

Actomyosin machinery endows cells with contractility at a single cell level. However, at a tissue scale, cells can show either contractile or extensile behaviour based on the direction of pushing or pulling forces due to neighbour interactions or substrate interactions. Previous studies have shown that a monolayer of fibroblasts behaves as a contractile system1 while a monolayer of epithelial cells2,3 or neural crest cells behaves as an extensile system.4 How these two contradictory sources of force generation can coexist has remained unexplained. Through a combination of experiments using MDCK (Madin Darby Canine Kidney) cells, and in-silico modeling, we uncover the mechanism behind this switch in behaviour of epithelial cell monolayers from extensile to contractile as the weakening of intercellular contacts. We find that this switch in active behaviour also promotes the buildup of tension at the cell-substrate interface through an increase in actin stress fibers and higher traction forces. This in turn triggers a mechanotransductive response in vinculin translocation to focal adhesion sites and YAP (Yes-associated protein) transcription factor activation. Our studies also show that differences in extensility and contractility act to sort cells, thus determining a general mechanism for mechanobiological pattern formation during cell competition, morphogenesis and cancer progression.

biophysics↗

bioRxiv2020

Adhesion-mediated heterogeneous actin organization governs apoptotic cell extrusion

Apoptotic extrusion is crucial in maintaining epithelial homeostasis and has implications in diseases of epithelial tissues. Current literature supports that epithelia respond to extrusion to maintain their integrity by the formation of a supracellular actomyosin ring (purse-string) in the neighbors that encompasses the dying cells. However, little is known about whether other types of actin structures could contribute to extrusion as well as how forces generated by mechanosensitive proteins in the cells are integrated. Here, we found that during extrusion, a heterogeneous actin network composed of lamellipodia protrusions and discontinuous actomyosin cables, was reorganized in the neighboring cells and was the main factor driving extrusion forwards. The early presence of basal lamellipodia protrusion participated both in basal sealing of the extrusion site and in orienting the actomyosin purse-string at the later stage of extrusion. These sequential events are essential in ensuring a successful extrusion in apicobasal direction. The co-existence of these two mechanisms is determined by the interplay between the cell-cell and cell-substrate adhesions. A theoretical model integrates the role of these cellular mechanosensitive components to explain why a dual-mode mechanism, which combined lamellipodia protrusion and purse-string contractility, leads to more efficient extrusion than a single-mode mechanism. We anticipate that our approach will be useful to provide mechanistic insight into epithelial homeostasis, morphogenetic events and tumorigenesis.

cell biology↗

bioRxiv2020

Endosomal spatio-temporal modulation of the cortical RhoA zone conditions epithelial cell organization

At the basis of cell shape and behavior, actomyosin organization and force-generating property are widely studied, however very little is known about the regulation of the contractile network in space and time. Here we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility, and we show that it is required for the maturation of stress fibers and frontrear polarity acquisition at the single cell level. There, EpCAM ensures the remodeling of a transient active RhoA zone in the cortex of spreading epithelial cells. GTP-RhoA follows the endosomal pathway mediated by Rab35 and EHD1, where it co-evolves together with EpCAM. In fact, EpCAM balances GTP-RhoA turnover in order to tune actomyosin remodeling for cell shape, polarity and mechanical property acquisition. Impairment of GTP-RhoA endosomal trafficking either by EpCAM silencing or Rab35 / EHD1 mutant expression prevents correct myosin-II activity, stress fiber formation, and ultimately cell polarization. Collectively, this work shows that the coupling of EpCAM/RhoA co-trafficking to actomyosin rearrangement is critical for spreading, and advances our understanding of how biochemical and mechanical properties can be coupled for cell plasticity.

cell biology↗

bioRxiv2019

Emergence of single cell mechanical behavior and polarity within epithelial monolayers drives collective cell migration

The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis1,2. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions3,4. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation5-7. Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations5,6,8,9 can arise from single cell behavior through a sustained memory of cell polarity.

biophysics↗

bioRxiv2019

Desmosomal coupling in apoptotic cell extrusion

The mechanical coupling of epithelia enables coordination of tissue functions and collective tissue movements during different developmental and physiological processes. This coupling is ensured by cell-cell junctions, including adherens junctions (AJs) and desmosomal junctions (DJs) [1, 2]. During apoptosis, or programmed cell death, a dead cell is expelled from the tissue by coordinated processes between the dying cell and its neighbors. Apoptotic cell extrusion is driven by actomyosin cable formation and its contraction, and lamellipodial crawling of the neighboring cells (Fig. S1A-A, Movie S1) [3-6]. Throughout cell extrusion, the mechanical coupling of epithelia needs to be maintained in order to preserve tissue homeostasis [3]. Although much is known about the regulation of AJs in apoptotic cell extrusion [6-9], the role and dynamics of DJs during this process remains poorly understood. Here, we show that DJs stay intact throughout and are crucial for apoptotic cell extrusion. Pre-existing DJs between the apoptotic cell and neighboring non-dying cells remain intact even during the formation of de novo DJs between non-dying cells, suggesting that the neighboring cells possess two DJs in the middle of apoptotic cell extrusion. We further found that an actomyosin cable formed in the vicinity of DJs upon apoptosis, and subsequently deviated from DJs during its constriction. Interestingly, the departure of the actomyosin cable from DJs coincided with the timing when DJs lost their straightness, suggesting a release of junctional tension at DJs, and a mechanical coupling between DJs and actomyosin contractility. The depletion of desmoplakin, which links desmosomes and intermediate filaments, resulted in defective apical contraction and an inability to form de novo DJs, leading to a failure of apoptotic cell extrusion. Our study provides a framework to explain how desmosomes play pivotal roles in maintaining epithelial sheet integrity during apoptotic cell extrusion.\n\nO_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC=\"FIGDIR/small/816280v1_figs1.gif\" ALT=\"Figure 1\">\nView larger version (49K):\norg.highwire.dtl.DTLVardef@454179org.highwire.dtl.DTLVardef@cd7e75org.highwire.dtl.DTLVardef@1295ed1org.highwire.dtl.DTLVardef@1102c8f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure S1:C_FLOATNO Desmosomal junctions at the interface during apoptotic extrusion. A) Confocal images showing actin (Lifeact-Ruby) dynamics during apoptotic cell extrusion in both apical (top panel) and basal (bottom panel) sections of the neighboring cell. Blue-filled and open arrows show the actomyosin cable (top panel) and the lamellipodia (bottom panel) respectively. A) Illustration showing the definition of interface ROI during extrusion. A) Graphs plotting normalized apoptotic cell area (black) and actin (blue) intensity at the dying cell interface during extrusion (n=5 independent experiments). B) Confocal images showing the localization of overexpressed Dsg-FLAG-EGFP (green) and endogenous desmoplakin (red) in MDCK monolayers (n=3 independent staining experiments). B) Magnified images of the white-filled and dotted boxes from B showing co-existence of dotted and continuous junctional clusters in the cell. C) Confocal images showing mCherry-E-cadherin dynamics at the interface during extrusion. C) Graphs plotting normalized apoptotic cell area (black) and E-Cadherin (blue) intensity during constriction (n=5 independent experiments). D) Confocal images showing desmoglein 2 and membrane (mCherry-delta-PH) dynamics in both apical (top panel) and basal (bottom panel) sections of the cell. White arrows (bottom panel) highlights formation of de novo junctions (n=8 independent experiments). E) Confocal images of endogenous desmosomes in the apical (top panel) and basal (bottom panel) sections during different phases of cell extrusion. Yellow and white arrows indicate pre-existing and de novo junctions, respectively (n=182 extrusion events from 7 independent experiments). F) Confocal images showing desmosome (green), killer red (mem-Killer red, cyan) and caspase 3 (magenta) before and after photo-irradiation. White arrow denotes extent of the dying cell. F) Confocal images showing desmosome dynamics (black) in the apical (top panel) and basal (bottom panel) sections of the neighboring cell. Red and blue arrows highlight pre-existing and de novo junctions respectively. F) Transverse view of a neighboring cell showing DJ dynamics after apoptosis induction through Killer Red. Red and blue arrows indicate pre-existing and de novo junctions respectively (n=4 independent experiments). The data from A and C represent mean {+/-}SEM. Scale bars, 10 m except C (5 m).\n\nC_FIG

cell biology↗

bioRxiv2019

Caveolae set levels of epithelial monolayer tension to eliminate tumor cells

Mechanical tension governs epithelial morphogenesis and homeostasis, but its regulation remains poorly understood. Tension is commonly contractile, arising when the actomyosin cortices of cells are mechanically coupled together by cadherin adhesion. Here we report that caveolae control levels of epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) in the surrounding epithelium, but not in the oncogene-expressing cells, blocked extrusion leading to the retention and proliferation of transformed cells within the monolayer. Tensile stress was aberrantly elevated in CAV1-depleted monolayers due to elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) causing increased recruitment of the formin, FMNL2. Oncogenic extrusion was restored to CAV1-deficient monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, by controlling lipid signalling to the actin cytoskeleton, caveolae regulate mechanical tension for epithelial homeostasis.

cell biology↗

bioRxiv2019

Myosin II isoforms play distinct roles in adherens junction biogenesis

Adherens junction (AJ) assembly under force is essential for many biological processes like epithelial monolayer bending, collective cell migration, cell extrusion and wound healing. The acto-myosin cytoskeleton acts as a major force-generator during the de novo formation and remodelling of AJ. Here, we investigated the role of myosinII isoforms in epithelial junction assembly. Myosin IIA (NMIIA) and Myosin IIB (NMIIB) differentially regulate biogenesis of adherens junction through association with distinct actin networks. Analysis of junction dynamics, actin organization, and mechanical forces of control and knockdown cells for myosins revealed that NMIIA provides the mechanical tugging force necessary for cell-cell junction reinforcement and maintenance. NMIIB is involved in E-cadherin clustering, maintenance of a branched actin layer connecting E-cadherin complexes and perijunctional actin fibres leading to the building-up of anisotropic stress. These data reveal unanticipated complementary functions of NMIIA and NMIIB in the biogenesis and integrity of AJ.

cell biology↗

bioRxiv2018

Sustained oscillations of epithelial cell sheets

Morphological changes during development, tissue repair, and disease largely rely on coordinated cell movements and are controlled by the tissue environment. Epithelial cell sheets are often subjected to large scale deformation during tissue formation. The active mechanical environment in which epithelial cells operate have the ability to promote collective oscillations, but how these cellular movements are generated and relate to collective migration remains unclear. Here, combining in vitro experiments and computational modelling we describe a novel mode of collective oscillations in confined epithelial tissues where the oscillatory motion is the dominant contribution to the cellular movements. We show that epithelial cells exhibit large-scale coherent oscillations when constrained within micro-patterns of varying shapes and sizes, and that their period and amplitude are set by the smallest confinement dimension. Using molecular perturbations, we then demonstrate that force transmission at cell-cell junctions and its coupling to cell polarity are pivotal for the generation of these collective movements. We find that the resulting tissue deformations are sufficient to trigger mechanotransduction within cells, potentially affecting a wide range of cellular processes.

biophysics↗

bioRxiv2018

Enhanced cell-cell contact stability upon Fibroblast Growth factor Receptor/N-cadherin cross-talk

N-cadherin adhesion has been reported to enhance cancer and neuronal cell migration either by mediating actomyosin-based force transduction or initiating Fibroblast Growth Factor Receptor (FGFR)-dependent biochemical signalling. Here we show that FGFR1 reduces N-cadherin-mediated cell migration. Both proteins are co-stabilised at cell-cell contacts through direct interaction. As a consequence, cell adhesion is strengthened, limiting the migration of cells on N-cadherin. Both the inhibition of migration and the stabilisation of cell adhesions require the FGFR activity stimulated by N-cadherin engagement. FGFR1 stabilises N-cadherin at the cell membrane through a pathway involving Src and p120. Moreover, FGFR1 stimulates the anchoring of N-cadherin to actin. We found that the migratory behaviour of cells depends on an optimum balance between FGFR-regulated N-cadherin adhesion and actin dynamics. Based on these findings we propose a positive feedback loop between N-cadherin and FGFR at adhesion sites limiting N-cadherin-based single cell migration.

cell biology↗

bioRxiv2018

Local Contractions Test Rigidity of E-Cadherin Adhesions

E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-cell contacts in tissues. Since epithelial cells respond to rigidity and tension in the tissue through E-cadherin, there must be active processes that test and respond to the mechanical properties of these adhesive contacts. Using sub-micrometer, E-cadherin-coated PDMS pillars, we find that cells generate local contractions between E-cadherin adhesions and pull to a constant distance for a constant duration, irrespective of pillar rigidity. These cadherin contractions require non-muscle myosin IIB, tropomyosin 2.1, -catenin and binding of vinculin to -catenin; plus, they are correlated with rigidity-dependent cell spreading. Without contractions, cells fail to spread to different areas on soft and rigid surfaces and to maintain monolayer integrity. We further observe that cadherin contractions enable cells to test myosin IIA-mediated tension of neighboring cells, and sort out myosin IIA-depleted cells. Thus, we suggest that epithelial cells test and respond to the mechanical characteristics of neighboring cells through cadherin contractions.

cell biology↗

bioRxiv2017

Collective cell migration without proliferation: density determines cell velocity and wave velocity

Collective cell migration contributes to morphogenesis, wound healing or tumor metastasis. Culturing epithelial monolayers on a substrate enables to quantify such tissue migration. By using narrow strips, we stabilise the front shape; by inhibiting cell division, we limit density increase and favor steady migration; by using long strips, we observe a confined cell monolayer migrating over days. A coherent collective movement propagates over millimeters; cells spread and density decreases from the monolayer bulk toward the front. Cell velocity ([~]micrometer per minute) increases linearly with cell radius, and does not depend explicitly on the distance to the front. Over ten periods of backwards propagating velocity waves, with wavelength [~]millimeter, are detected with a signal-to-noise ratio enabling for quantitative spatio-temporal analysis. Their velocity ([~]ten micrometers per minute) is ten times the cell velocity; it increases linearly with the cell radius. Their period ([~]two hours) is spatially homogeneous, and increases with the front density. When we inhibit the formation of lamellipodia, cell velocity drops while waves either disappear, or have a smaller amplitude and slower period. Our phenomenological model assumes that both cell and wave velocities are related with the activity of lamellipodia, and that the local stretching in the monolayer bulk modulates traction stresses. We find that parameter values close to the instability limit where waves appear yield qualitative and quantitative predictions compatible with experiments, including the facts that: waves propagate backwards; wave velocity increases with cell radius; lamellipodia inhibition attenuates, slows down or even suppresses the waves. Together, our experiments and modelling evidence the importance of lamellipodia in collective cell migration and waves.

biophysics↗

bioRxiv2017

Force-Dependent Binding Of Vinculin To α-Catenin Regulates Cell-Cell Contacts Stability And Collective Cell Behavior

The shaping of a multicellular body and repair of adult tissues require fine-tuning of cell adhesion, cell mechanics and intercellular transmission of mechanical load. Adherens junctions (AJs) are the major intercellular junctions by which cells sense and exert mechanical force on each other. However, how AJs adapt to mechanical stress and how this adaptation contributes to cell-cell cohesion and eventually to tissue-scale dynamics and mechanics remains largely unknown. Here, by analyzing the tension-dependent recruitment of vinculin, -catenin and F-actin as a function of stiffness, as well as the dynamics of GFP-tagged wild-type and mutated -catenins, altered for their binding capability to vinculin, we demonstrate that the force-dependent binding of vinculin stabilizes -catenin and is responsible for AJ adaptation to force. Challenging cadherin complexes mechanical coupling with magnetic tweezers, and cell-cell cohesion during collective cell movements, further highlight that tension-dependent adaptation of AJs regulates cell-cell contact dynamics and coordinated collective cell migration. Altogether, these data demonstrate that the force-dependent -catenin/vinculin interaction, manipulated here by mutagenesis and mechanical control, is a core regulator of AJ mechanics and long-range cell-cell interactions.\n\nSummary statementCombining cell biology and biomechanical analysis, we show here that the coupling between cadherin complexes and actin trough tension-dependent -catenin/vinculin association is regulating AJ stability and dynamics as well as tissue-scale mechanics.

cell biology↗