The importance of intermediate filaments in the shape maintenance of myoblast model tissues
- Laboratoire Matière et Systèmes Complexes, UMR 7057, Université Paris Cité and CNRS, France
- Laboratoire Biologie Fonctionnelle et Adaptative, UMR 8251, Université Paris Cité and CNRS, France
Figures
Figure 1 with 4 supplements
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 …
Figure 1—figure supplement 1
Metabolic activity for unlabelled (CTL) and labelled cells ([Fe] = 2–16 mM and 2 hr incubation time), 2 hr (D0), and 1 day (D1) after nanoparticle incorporation.
To assess metabolic activity, the metabolic test Alamar Blue was used. Fluorescence was measured at nm and nm. Values are interpreted relative to control values (unlabelled cells in complete …
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Figure 1—figure supplement 1—source data 1
Source data of the Alamar-Blue test values for the different magnetic labelling conditions reported in Figure 1—figure supplement 1.
Measurement of the fluorescence ( nm and nm) after 1h30 incubation with the Alamar blue solution.
- https://cdn.elifesciences.org/articles/76409/elife-76409-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Immunofluorescence images of C2C12 spheroid cryosections to confirm non-hypoxic and non-apoptotic conditions in spheroids.
(a) DAPI is shown in blue and HIFα in green. HIFα is almost absent from the images. (b) DAPI is shown in blue and cleaved caspase-3 in green. Only 2% of cells show a positive signal to cleaved …
Figure 1—figure supplement 3
Extraction of experimental volume, width, and height of the spheroid.
The experimental volume V is calculated from the radius R of the spheroid at t0. The experimental width w (in green) and height h (in blue) are measured from the spheroid equilibrium profile. All …
Figure 1—figure supplement 4
Relative height of the aggregate as a function of time for control, latrunculin A (LatA), EGTA, and (±)-blebbistatin ((±)-Bleb) conditions.
At s, the magnet is approached and the height of the spheroid is monitored over time. After 10 min of flattening, the equilibrium shape is reached and the height of the spheroid remains stable …
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Figure 1—figure supplement 4—source data 1
Source data of the evolution of the height of multicellular aggregates presented on Figure 1—figure supplement 4.
The dynamic of flattening process is reported for 5 different aggregates.
- https://cdn.elifesciences.org/articles/76409/elife-76409-fig1-figsupp4-data1-v2.xlsx
Figure 2 with 3 supplements
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 …
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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
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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
Figure 2—figure supplement 1
Immunofluorescence images of C2C12 spheroid cryosections for control conditions (a), 0.25 µM latrunculin A (b), and 2.5 mM EGTA (c).
DAPI is shown in blue, pan-cadherins are in green, and F-actin in red. Scale bars: 200 µm and 40 µm for zoomed images.
Figure 2—figure supplement 2
Young’s modulus of C2C12 spheroids as a function of their surface tension.
Young’s modulus and surface tension are proportional across all the conditions tested for C2C12 spheroids. Values are represented in logarithmic scale.
Figure 2—figure supplement 3
Immunofluorescence images of C2C12 spheroid cryosections for 160 µM (+)-blebbistatin (a) and 160 µM (±)-blebbistatin (b).
DAPI is shown in blue, pan-cadherins are in green, and F-actin in red. Scale bars: 200 µm and 40 µm for zoomed images.
Figure 3 with 1 supplement
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 …
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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
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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
Figure 3—figure supplement 1
Comparison between local angles measured on spheroid cryosections or 3D images for C2C12 WT spheroids.
Local angles were measured on the surface of the spheroids for spheroids for each type of measurement. Measured angles on cryosections have a median of 145°, and measured angles in 3D images have …
Figure 4 with 3 supplements
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., …
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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.xlsx
Figure 4—figure supplement 1
Desmin organization Immunofluorescence images of desmin on adherent cells of control A21V, desWT-Cl29, and desD399Y-Cl26 cell lines for no heat shock (HS) or 2 hr HS.
Hoechst signal (nuclei) is shown in blue, and desmin is shown in green. Scale bar: 20 µm.
Figure 4—figure supplement 2
Immunofluorescence images of C2C12 spheroid cryosections of desWT-Cl29 (a, c) and desD399Y-Cl26 cells (b, d) for no heat shock (HS) (a, b) or 2 hr HS (c, d).
DAPI is shown in blue, and Myc is shown in green. Scale bars: 200 µm and 40 µm for zoomed images.
Figure 4—figure supplement 3
Surface tension and Young’s modulus of A21V spheroids (a), of desWTCl29 spheroids (b) and desD399Y-Cl26 (c) with no heat shock (HS), HS 30 min or HS 2 hr.
Figure 5 with 1 supplement
Geometrical analysis of cells at the aggregate surface for C2C12 A21V, desWT-Cl29, and desD399Y-Cl26 spheroids.
(a) Profile surface roughness parameter Rq (root-mean-squared) in each condition for at least spheroids. (b) Local contact angle between cells at the surface measured in each condition for at …
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Figure 5—source data 1
Source data of the roughness of multicellular aggregates for control A21V, desWT-Cl29, and desD399Y-Cl26 cells presented in Figure 5a.
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-fig5-data1-v2.xlsx
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Figure 5—source data 2
Source data of the local contact angles measured for control A21V, desWT-Cl29, and desD399Y-Cl26 cells presented in Figure 5b, e and f.
Local contact angles of cells within an aggregate are manually measured, and the distribution of the measured local contact angles is given. From the mean surface tension, both tension at the cell–medium and cell–cell medium are deduced for each cell type.
- https://cdn.elifesciences.org/articles/76409/elife-76409-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
Quantification of phospho-myosin distribution in the spheroids.
(a–c) Immunofluorescence images of C2C12 WT (a) or A21V, desWT-Cl29, and desD399Y-Cl26 (c) spheroid cryosections in the different experimental conditions. Nuclei are shown in blue, and …
Figure 6
