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• W4313031109 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Liquid and elastic behaviours of tissues drive their morphology and response to the environment. They appear as the first insight into tissue mechanics. We explore the role of individual cell properties on spheroids of mouse muscle precursor cells and investigate the role of intermediate filaments on surface tension and Young’s modulus. By flattening multicellular myoblast aggregates under magnetic constraint, we measure their rigidity and surface tension and show that they act as highly sensitive macroscopic reporters closely related to microscopic local tension and effective adhesion. Shedding light on the major contributions of acto-myosin contractility, actin organization, and intercellular adhesions, we reveal the role of a major component of intermediate filaments in the muscle, desmin and its organization, on the macroscopic mechanics of these tissue models. Implicated in the mechanical and shape integrity of cells, intermediate filaments are found to be crucial to the mechanics of unorganized muscle tissue models even at an early stage of differentiation both in terms of elasticity and surface tension. Editor's evaluation This important work studied the determinants of key physical properties of multicellular assemblies using magnetic flattening of spheroids. The key and convincing result is that intermediate filaments could also be implicated in the setting of the elastic properties of these assemblies, shedding light on this central cellular component and how their modifications could be important to the understanding of some pathologies. https://doi.org/10.7554/eLife.76409.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Tissue-forming cells interact with each other and with their environment (Barone and Heisenberg, 2012; Humphrey et al., 2014; Indana and Chaudhuri, 2021) giving rise to interesting viscoelastic fluid behaviours (Lecuit and Lenne, 2007) that are determinant both in epithelia (Mongera et al., 2018) and 3D tissue-forming systems (Maître et al., 2012). Physical properties such as surface tension (Ehrig et al., 2019; Harmand et al., 2021) and viscosity (Stirbat et al., 2013) can be introduced to predict tissue organization and shape (Etournay et al., 2015). Global mechanical characteristics of tissues emerge from individual cell components and their interplay (Jakab et al., 2008; Dolega et al., 2021; Grosser et al., 2021), but whether they could be good reporters of individual cell behaviour and global organization is still unknown. Multicellular aggregates (Kalantarian et al., 2009; Stirbat et al., 2013) prove to be powerful tools to apprehend fundamental biological processes as morphogenesis (Costa et al., 2016), development (Birey et al., 2017), and tumorigenesis (Montel et al., 2011; Gunti et al., 2021). They are able to mimic various biological phenomena (Nikolaev et al., 2020), and as model systems, are easier to use and monitor than in vivo tissues (Bassi et al., 2021). Combining simplicity, reproducibility, and biological significance, they are a system of choice both for biophysics and computation (Gonzalez-Rodriguez et al., 2012; Martin and Risler, 2021; Ackermann et al., 2021). Myoblasts are widely studied cells to understand myogenesis because of their interest in myopathy modelling (Smoak et al., 2019) and drug testing (Zhuang et al., 2020). Muscle cell mutations implicated in numerous diseases have been extensively studied starting from symptoms to molecular origins identification (Batonnet-Pichon et al., 2017; Jungbluth et al., 2018). Among these mutations, the ones concerning intermediate filaments are very important (Dutour-Provenzano and Etienne-Manneville, 2021). Intermediate filament network is essential in muscle development, provides mechanical integrity to the cell (Chang and Goldman, 2004), and plays a major role in the dynamic response to mechanical stimulation (Charrier et al., 2016). Desmin, as a major component of intermediate filaments specifically expressed in smooth, skeletal, and cardiac muscles, presents some mutations associated to muscle defects and myopathies. However, the early effects of these mutations in tissues are unclear due to a lack of in vitro biomimetic muscle systems (Hofemeier et al., 2021). To address these limitations, we focus on mouse myoblast cells (C2C12) to test the sensitivity of 3D unorganized early-stage muscle tissue models to individual cell modifications. C2C12 are adhesive and highly contractile cells (unsurprisingly regarding their function). Their high assumed surface tension makes them challenging to characterize. We designed an integrated sensitive magnetic tensiometer (Mazuel et al., 2015) to form stimulable myoblast-derived tissues and measure their surface tension and elasticity. In this study, we explore the interplay between macroscopic properties of model muscle tissues and the molecular or cellular processes. We investigate how surface tension and Young’s modulus represent appealing tools to determine from the tissue scale the microscopic properties. While actin and cadherins are implicated in the surface tension of multicellular aggregates or embryos (Foty and Steinberg, 2005), the role of intermediate filaments in tissue shape maintenance has never been identified. Mutations in intermediate filaments severely hinder individual cell nanomechanical properties (Herrmann et al., 2007), and their network supports the shape of individual cells (Goldman et al., 1996) and withstands applied constraints. Looking at the interplay between their organization and tissue shape maintenance or the tissue elasticity is thus primordial, especially in the context of muscle. We look at cells expressing mutated desmin and exhibiting organization defects of the desmin cellular network to shed light on the crucial role of intermediate filaments on muscle tissue model global mechanics at an early stage of differentiation. Results Magnetic tensiometer for multicellular aggregates C2C12 cells are labelled with superparamagnetic nanoparticles without altering their biological capacities (Van de Walle et al., 2020a; Van de Walle et al., 2020b) nor inducing hypoxia or apoptosis (Figure 1—figure supplements 1 and 2). It is then possible to organize and stimulate them at will using external magnets. Magnets first drive cells in agarose moulds to create spheroids of controlled size (Mazuel et al., 2015) and content as inhibitors or reagents can be added at this stage (Figure 1a). Cohesive spheroids are obtained within 12 hr (Figure 1b) and their side profile is imaged (Figure 1c). Multicellular spheroids are fulfilled with cohesive cells that organize to form a tissue model with rounded cells at the core of the aggregate having cortical actin and more elongated cells at the periphery having contractile actin network. Their profile is recorded while a magnet is approached (Figure 1—figure supplements 3 and 4). Assuming that a multicellular cohesive aggregate can be modelled as a continuous elastic medium (as supported by confocal imaging, Figure 1b), surface tension and magnetic forces in volume compete to determine the equilibrium shape (Kalantarian et al., 2009) while Young’s modulus is at stake for the contact area (Mazuel et al., 2015; Figure 1c, Figure 1—figure supplement 3). The height, the width of the aggregate and the ending points of the contact zone are pointed in the Tensio X application (Figure 1—figure supplement 3), a dedicated MATLAB interface we developed to fit the obtained profile. The elasticity and the capillary parameter c=Mv⁢g⁢r⁢a⁢d⁢(B)γ (Mv, γ, and B denote the magnetic moment per unit of volume, the spheroid surface tension, and the magnetic field, respectively) (Kalantarian et al., 2009) are extracted. By knowing the magnetic volume moment, the tissue surface tension is easily deduced from c. The error measurement on the deformations is around 2–5 µm, leading to a precision in the range of 5–20% for surface tension and 5–10% for the elasticity of C2C12 multicellular spheroids. In this regard, it should be noted that the surface tension of C2C12 spheroids is two orders of magnitude greater than that of classically studied cells such as F9 cells, for example. The error increases with surface tension because the spheroids are less deformed, which makes C2C12 cells challenging to characterize. This error is smaller than the inherent distribution measured over different aggregates (Figure 2a and b). Figure 1 with 4 supplements see all Download asset Open asset Magnetic tensiometer integrated measurement set-up. (a) Schematic of the magnetic moulding process. A network of calibrated size steel beads deposited over cylindrical magnets is embedded in heated 2% liquid agarose (1). After agarose gelling, beads are removed creating a semi-spherical mould (2). Magnetically labelled cells are seeded in these non-adhesive-treated moulds. Magnets placed below each mould drive the cells inside the moulds (3). Due to high local cell density, cell–cell contacts develop to form cohesive multicellular aggregates within 12 hr (4). (b) Left panel: confocal image of a multicellular aggregate at 150 µm from the top of the aggregate. Actin (red) and nuclei (blue) are labelled. Scale bar: 50 µm. Right panels: 75-µm-long zooms for cells at the interface (up) or in the centre of the multicellular aggregate (down). The organization of actin depends on the location of the cell within the aggregate. While it is mainly cortical for cells in the centre, actin is contractile at the periphery of the aggregate to maintain its shape. Scale bar: 25 µm. (c) Schematic of the magnetic force tensiometer set-up. A temperature-regulated tank (37°C) is sealed with a non-adhesive glass slide to ensure non-wetting conditions for the multicellular spheroid formed by magnetic moulding (‘Methods’ and a). A cylindrical neodymium permanent magnet (6 mm diameter and height, 530 mT, grad(B)=170T.m−1) is positioned underneath and approached with respect to a lifting stage. The tank is filled with a transparent culture medium, and the aggregate side profile is monitored with a camera. (d) Representative pictures of C2C12 spheroid profiles. Top and bottom pictures show, respectively, a C2C12 spheroid before the magnet approach (t0) and under magnetic flattening when the equilibrium shape is reached (t∞). The dynamics is shown in Figure 1—figure supplement 4. The spheroid surface tension is measured by fitting the aggregate shape with Laplace profile (red line) while the elastic modulus is extracted from the radius of the contact zone L (green arrow) using Hertz theory as described in Mazuel et al., 2015; Figure 1—figure supplement 3. In this picture, γ=21mN/m and E=100⁢Pa. Scale bar: 200 µm. Figure 2 with 3 supplements see all Download asset Open asset Co-action of cortical tension and intercellular adhesions in multicellular spheroid surface tension and Young’s modulus. (a, b) Variation of surface tension (a) and Young’s modulus (b) of C2C12 spheroids for 2.5 mM EGTA (calcium chelator), 0.15 µM and 0.25 µM latrunculin A (actin disruptor). Floating bars represent min to max variations, and the midline indicates the median. (c) Representative confocal images of acto-myosin organization for cells at the periphery of an untreated multicellular aggregate. Nuclei (blue), F-actin (red) and phospho-myosin (green) are labelled. Actin and myosin networks mostly colocalize and form a contractile radial organization. Scale bar: 20 µm. (d) Representative confocal image of acto-myosin organization for cells at the periphery of a multicellular aggregate treated with 160 µM (±)-blebbistatin. Nuclei (blue), F-actin (red), and phospho-myosin (green) are labelled. Myosin network is almost disrupted compared to actin network. Scale bar: 20 µm. (e) Representative pictures of C2C12 spheroids under magnetic flattening at equilibrium for (±)-blebbistatin concentration ranging from 0 to 160 µM are given for comparison. Scale bar: 200 µm. Variations of both surface tension (f) and Young’s modulus (g) with (±)-blebbistatin concentrations are reported. Inhibition curves are fitted for the surface tension (red curve) and the Young’s modulus (green curve) with a dose–response providing an I⁢C50 value for the (±)-blebbistatin of 10 ± 3 µM and 7 ± 5 µM, respectively, corresponding to an I⁢C50 for the (-) active enantiomer of blebbistatin around 6 ± 2 µM and 4 ±3 µM, respectively, as the ratio of the active/negative form is around 50–60%. Figure 2—source data 1 Source data of surface tension and Young’s modulus measurements for control cell aggregates, EGTA, or latrunculin A-treated cell aggregates reported in Figure 2a and b. Both surface tension and Young’s modulus were extracted from aggregate profiles using TensioX application. Means, medians. and standard deviations were extracted. https://cdn.elifesciences.org/articles/76409/elife-76409-fig2-data1-v2.xlsx Download elife-76409-fig2-data1-v2.xlsx Figure 2—source data 2 Source data of the surface tension and Young’s modulus measurements for control cell aggregates and blebbistatin-treated cell aggregates reported in Figure 2f and g. ±-Blebbistatin was used at different concentrations. Both surface tension and Young’s modulus were extracted from the aggregates profiles using TensioX application. Means, medians, and standard deviations were extracted for each condition and compared. https://cdn.elifesciences.org/articles/76409/elife-76409-fig2-data2-v2.xlsx Download elife-76409-fig2-data2-v2.xlsx Relation between macroscopic properties and molecular or cellular characteristics: A multi-contribution pattern Numerous molecular origins of spheroid surface tension are identified. While differential adhesion hypothesis was first evidenced by Steinberg, 1963 and related to the level of cadherins (Foty and Steinberg, 2005), the role of actin cortex was pointed out in individual cells (Chugh et al., 2017), spheroids (Stirbat et al., 2013), or embryogenesis (Heer and Martin, 2017), evidencing a differential interfacial tension hypothesis combining the influence of adhesion and cell surface tension (Manning et al., 2010). By comparing the energy of cells in the core of the aggregate to the one at the interface, the surface tension is given by (1) γ=TCM−12(2TCC−JCC) where TC⁢M, TC⁢C, and JC⁢C denote the cortical tension at the cell–medium (CM) or at the cell–cell (CC) interface and the intercellular surface adhesion energy (JCC>0), respectively (Stirbat et al., 2013). We explore this multi-parameter influence by looking at CC contacts inhibition and changes in actin structure or acto-myosin contractility (Figure 2). Intercellular adhesion is mediated by multiple CC adhesion proteins, among which cadherins play a key role. EGTA, as a calcium chelator, reduces the efficiency of this homophilic adhesion and modulates CC adhesion strength. In multicellular aggregates, EGTA has an impact on the acto-myosin network (Figure 3f, Figure 2—figure supplement 1). By reducing CC contacts, it impairs the formation of the contractile network of acto-myosin at the periphery of the aggregate. At the single-cell level, F-actin becomes mainly cortical whatever the cell location inside the aggregate is. It may be related to a lack of strong enough CC adhesions to maintain actin network and a co-regulation of actin and cadherin tension. EGTA addition leads to a more than fivefold decrease in both Young’s modulus and surface tension (Figure 2a and b). The relationship between adhesion bond energy and tissue surface tension (Foty and Steinberg, 2005) is thus tested. An apparent proportionality between surface tension and Young’s modulus is obtained (Figure 2—figure supplement 2), reminiscent of the one observed between surface tension and elasticity of the whole aggregate (itself related to cortical tension) through co-regulation mechanisms (Yu et al., 2018). Figure 3 with 1 supplement see all Download asset Open asset Geometrical analysis of cells at the aggregate surface. (a) Immunofluorescence images of cryosections of multicellular aggregates obtained by magnetic moulding with different conditions. Control cells are compared to aggregates produced with 160 µM (+)-blebbistatin (inactive enantiomer), 160 µM (±)-blebbistatin (mixture of active and inactive enantiomers), 2.5 mM EGTA or 0.25 µM latrunculin A. DAPI is shown in blue, pan-cadherin in green, and F-actin in red. Scale bar: 50 µm. (b) Profile surface roughness parameter Rq (root-mean-squared) in each condition for at least N=3 spheroids. (c) Schematic of two neighbouring cells with a local contact angle α and the respective tension at the cell–medium interface TC⁢M and effective tension at the cell–cell contact 2⁢TC⁢C-JC⁢C. (d) Examples of immunofluorescence images of spheroids from which the local angles were measured. Nuclei are shown in blue and pan-cadherin in green. Scale bar: 10 µm. (e) Contact angle between cell surfaces measured in each condition for at least N=3 spheroids either on cryosections or 3D aggregates (Figure 3—figure supplement 1). (f) Examples of immunofluorescence images of the upper cells on spheroids obtained with different conditions. Nuclei are shown in blue, actin in red, and phospho-myosin in green. Colocalization of phospho-myosin and actin is in yellow. Scale bar: 25 µm. (g, h) Deduced values of the cell tension at the cell–medium interface (g) and of the effective adhesive tension at the cell–cell contact (h) in each condition. Figure 3—source data 1 Source data of the roughness of imaged multicellular aggregates in various conditions presented in Figure 3a. Roughness of the different multicellular aggregates was reported and compared. Aggregate contours were first extracted, then the roughness of the contour was calculated. Means, medians, and standard deviations are reported for each condition. https://cdn.elifesciences.org/articles/76409/elife-76409-fig3-data1-v2.xlsx Download elife-76409-fig3-data1-v2.xlsx Figure 3—source data 2 Source data of the local contact angles and the tension at the cell–medium and the cell–cell interfaces measured on multicellular aggregates in various conditions reported in Figure 3e, g and h. Local contact angles of cells within an aggregate are manually measured, and the distribution of the local contact angle is given. From the mean surface tension, both tension at the cell–medium and cell–cell medium are deduced. https://cdn.elifesciences.org/articles/76409/elife-76409-fig3-data2-v2.xlsx Download elife-76409-fig3-data2-v2.xlsx Latrunculin A disrupts the actin filaments by binding to actin monomers, thus precluding its polymerization (Coué et al., 1987) its addition gives some non-connected patches of actin filaments with a lack of long-range organization (Figure 3f, Figure 2—figure supplement 1). Young’s modulus, as well as surface tension, is dramatically decreased by its presence (Figure 2a and b). Besides, its action on macroscopic properties is dose-dependent as seen by the comparison between 0.15 µM (twofold decrease) and 0.25 µM (five- to sevenfold decrease) concentrations. Our results are consistent with the dependence previously noticed between surface tension and viscosity for latrunculin-treated cells (Jakab et al., 2008) and extend this dependence to stiffness. To further test the sensitivity of the magnetic tensiometer, we selected reagents with a wider accessible range still allowing cohesive spheroid formation (Figure 2—figure supplement 3). (-)-Blebbistatin inhibits contractility by blocking myosins. (±)-Blebbistatin is a cell-permeable mixed compound containing around 50–60% of the active negative enantiomer that acts as a selective, potent, and reversible inhibitor of non-muscle myosin II without affecting actin filaments assembly. In the multicellular aggregates environment, it impairs the activity of myosins reducing contractility (Figure 2c and d). Its inhibition potency is quantified in vitro by I⁢C50 values ranging from 2 to 7 µM depending on myosin type (Zhang et al., 2017). In our experiments, (±)-blebbistatin is used in a wide range of concentrations (0–160 µM) and leads to an increase in the aggregate deformation with increasing concentration (Figure 2e) while the inactive (+)-blebbistatin does not affect mechanical properties. Surface tension and Young’s modulus dramatically decrease with (-)-blebbistatin concentration (Figure 2f and g) with a decay of up to tenfold. The extracted I⁢C50 is around 6 ± 2 µM for surface tension and 4 ±3 µM for elasticity, reproducing the one obtained at the molecular level (as the ratio of the active negative enantiomer is around 50–60%). Correlation with geometrical analysis: Tensions at the interfaces By analogy with fluids, surface tension in multicellular aggregates arises from the energy difference between cells at the interface with the medium and cells surrounded by others. The shape of cells at the surface of multicellular aggregates can thus be used to relate tissue surface tension to cell tensions. Looking at cell surface morphology on multicellular aggregate cryosections (Figure 3a) upon the use of the different drugs, we are able to test the relation between surface cell shape and arrangement, on the one hand, and macroscopic surface tension, on the other. Latrunculin A and EGTA-treated aggregates show rounded cells at the interface while control cells flatten on the surface without extending over multiple cells. (±)-Blebbistatin-treated aggregates have an intermediate behaviour. To quantify these observations, we extracted both roughness of the profile and mean contact angle α between cells at the interface (Figure 3). They show similar variations. For control aggregates and (+)-blebbistatin aggregates, cells at the surface spread out, having a flat angle at the CC contacts and a small roughness length. Drug drastic effects can be noticed on EGTA and latrunculin A-treated aggregates: roughness increases (by 3 for the latrunculin A treated cells and by 2 for EGTA treated cells) while contact angles deviate from flat angle to get close to 100°. Overall, the surface tension variations are correlated with a change in morphology of the cells at the interface. As already noticed (Manning et al., 2010), high tissue surface tension usually appears as the hallmark of flattened cells and low roughness. However, the case of (±)-blebbistatin inhibitor shows that multiple parameters have to be considered as the low surface tension obtained with the myosin inhibitor does not lead to rounded cells at the interface. Tissue surface tension arises from a balance between cortical tension at the CM and adhesion and cortical tension at the CC interface (Equation 1; Manning et al., 2010; Stirbat et al., 2013). Tissue surface tension quantifies this interplay and not only the tension at the CM interface while being predominant. None of the considered aggregates shows elongated surface cells extending over multiple inner cells, meaning that the effective CC interfacial energy 2⁢TC⁢C-JC⁢C is smaller than the double cortical tension at the CM interface. In this configuration, differential interfacial tension hypothesis and more sophisticated models provide similar results (Manning et al., 2010), and the local mechanical equilibrium at the three phases cell/cell/medium contact line gives the relation (Stirbat et al., 2013) (2) 2TCMcos⁡(α2)=2TCC−JCC Both effective CC tension 2⁢TC⁢C-JC⁢C and cortical tension at the CM interface TC⁢M can then be deduced from surface tension and contact angle measurements: TCM=γ1−cos⁡α2;2TCC−JCC=2γcos⁡α21−cos⁡α2 First, we checked whether the tension at the CM interface is predominant in the value of surface tension. As noticed on the cryosections, EGTA and latrunculin A impact predominantly the tension at the CM interface with a reduction by a factor of around Figure 3g. By reducing CC adhesion, EGTA impairs the formation of contractile actin network at the frontier of the aggregate, and actin is mostly cortical (Figure 3f). By depolymerizing actin, latrunculin A reduces global cortical tension as well as the CC adhesion by weakening cadherin anchorage through actin. Feedback between adhesion molecules and the cytoskeleton is indeed abundant and explains the lower variations of effective CC tension upon latrunculin A and EGTA addition due to a possible compensation of the tension decrease by a decrease in adhesion (JC⁢C). Besides, blebbistatin has a significant effect on both effective adhesion and tension at the CM as it impacts neither intercellular adhesions nor the cytoskeleton structure but the ability of the cell cortex to contract. By decreasing tension at the cortex, this inhibitor reduces both TC⁢M and TC⁢C, thus affecting tension and effective CC adhesion in a more drastic way than latrunculin or EGTA. Hence, the magnetic tensiometer appears as a highly sensitive tool to look at tissue model mechanics and its relation with modifications at the cellular level. Role of intermediate filaments in macroscopic mechanical properties of muscular tissue models Desmin is an intermediate filament specific to muscle cells where it plays an essential role in maintaining mechanical integrity and elasticity (Even et al., 2017) at the single-cell level. It stands as a marker for muscular cell differentiation, but its role in tissue surface tension maintenance and elasticity has not been explored. Desmin mutations are involved in human diseases such as certain skeletal and cardiac myopathies (Goldfarb et al., 2004), characterized histologically by intracellular protein aggregates containing desmin. We focus on C2C12 myoblasts expressing desmin with the missense mutation D399Y (Segard et al., 2013). We use three cell lines: A21V cells, which are stably transfected with an empty vector and are control cells; desWT-Cl29 cells, which stably express exogenous wild-type (WT) desmin with a ratio of around 1:1 compared to endogenous desmin; and desD399Y-Cl26 cells, which stably express exogenous mutated desmin with a ratio of around 1:1 compared to endogenous desmin (Delort et al., 2019). Desmin overexpression and modification do not affect desmin organization of adherent cells in the absence of induced protein aggregation (Figure 4—figure supplement 1). Surface tension and Young’s modulus are not or hardly impacted by the overexpression of desmin (wild-type or mutated) (Figure 4f). Comparing A21V cells to desWT-Cl29, one can notice a slight decrease in elasticity (Figure 4f) but it is not measured for desD399Y-Cl26 cells. Besides, as the local contact angle and roughness are similar in all three cell types (Figure 5a and b), the deduced CM and effective CC tensions also have values which are not significantly different (Figure 5e and f). The three actin networks and the phospho-myosin networks (Figure 5—figure supplement 1) are also similar. Figure 4 with 3 supplements see all Download asset Open asset Effect of heat shock (HS) and protein aggregation of desmin on surface tension and Young’s modulus of C2C12 spheroids. (a) Representation of desmin with the missense mutation D399Y located in Rod Domain. The expressed exogenous mutated desmin is Myc-tagged at the N-terminus (adapted from Figure 1 from Segard et al., 2013). (b) Experimental procedure. Desmin expression was induced 24 hr after cell plating with doxycycline for 48 hr. Magnetically labelled cells (see ‘Methods’) experienced an HS for 0, 30, or 120 min before spheroids were moulded. Spheroid surface tension and Young’s modulus were measured 3 days after expression induction. (c) Immunofluorescence images of desWT-Cl29 (cells stably expressing exogenous desmin WT) and desD399Y-Cl26 cells (cells stably expressing exogenous mutated desmin) in 2D after 0, 30, or 120 min HS. DAPI is shown in blue and the Myc-tag in green. Scale bar: 20 µm. (d) Percentage of cells with desmin protein aggregates for each condition. Desmin protein aggregation increases for desD399Y-Cl26 cells with the duration of the HS while it remains stable around 2% for desWT-Cl29 cells. Mean values represented with respective standard deviations and at least three independent experiments for each condition. (e) Immunofluorescence images of multicellular aggregate cryosections of desWT-Cl29 or desD399Y-Cl26 cells with or without HS. DAPI is visible in blue and the Myc-tag in green. The results are reminiscent of the ones in 2D. DesD399Y-Cl26 spheroids exhibit sparse aggregation without HS, enhanced by 2 hr HS. Scale bar: 200 µm. See Figure 4—figure supplement 2. (f) Surface tension and Young’s modulus of A21V cells (control cells stably transfected with empty vector) spheroids compared with desWT-Cl29 and desD399Y-Cl26 cells to test for the influence of desmin overexpression. (g) Surface tension and Young’s modulus of A21V cells, desWT-Cl29, and desD399Y-Cl26 spheroids with an HS of 0 (no HS), 30 or 120 min. (f, g) At least three independent experiments for each condition and N=8 spheroids. Floating bars represent min to max variations, and the midline indicates the median. Figure 4—source data 1 Source data for the surface tension and Young’s modulus measurements of aggregates shown in Figure 4d, f and g. Both surface tension and Young’s modulus were extracted from the profile of aggregates made of either control A21V, desWT-Cl29, or desD399Y-Cl26 cells using TensioX application. Means, medians, and standard deviations were extracted for each cell type and compared. https://cdn.elifesciences.org/articles/76409/elife-76409-fig4-data1-v2.x" @default.
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• W4313031109 title "Editor's evaluation: The importance of intermediate filaments in the shape maintenance of myoblast model tissues" @default.
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