Figure 5. Differential contributions of NM II isoforms to cellular mechanoresponse.

Cells were cultured 3D micro-scaffolds composed of four non-adhesive walls, each with an inward directed protein-adhesive bar to guide cell attachment. Cells attach to the bars, form a cross-shaped morphology and pull the walls inwards. (A) Initial cellular tractions forces were determined by detaching the cell from the scaffold using trypsin/EDTA and measuring the corresponding average beam displacement as indicated in the plot. (B) Comparison of the initial forces of the different cell lines. Data for WT and NM IIA-KO have been reproduced from (B) of Hippler et al., 2020, therefore only the mean values are shown (originally published under the Creative Commons Attribution-Non Commercial 4.0 International Public License (CC BY-NC 4.0; https://creativecommons.org/licenses/by-nc/4.0/. Further reproduction of this panel would need to comply with the terms of this license)). No significant differences were observed between WT (mean value = 94 nN), NM IIB-KO (mean value = 112 nN), and NM IIC-KO cells (mean value = 110 nN). A significant decrease was observed for NM IIA-KO cells (mean value = 11 nN). (C) Illustration depicting the stretch-release cycle applied to the cells. (D-G) Examples of average beam displacements (corresponding to Figure 5—animations 5–8) are plotted as a function of time. The blue area depicts the time frame, in which the corresponding cell was stretched. (D) WT cells actively counteract the stretch and increase their contractile forces until reaching a plateau after ~ 30–40 min. After releasing the stretch, cellular contraction forces remained high, but decreased to the initial level after 20–30 min. (E) No cellular force response is observed, when applying the stretch-release cycle to NM IIA-KO cells, even after longer stretch periods. (F) NM IIB-KO cells increase their force after stretching and reach a plateau after 30–40 min. The force increase is lower compared to WT cells. After releasing the stretch, NM IIB-KO cells also reduce their forces until the initial set point is reached. (G) NM IIC-KO cells increase their force upon the stretch but do not relax to the initial setpoint within the observed timeframe. (H) The quantification shows that a force decrease after the stretch release was observed for 78% of the WT cells but only for 22% of the NM IIC-KO cells. (I) Comparison of the force increase of WT and NM II-KO cells, after being mechanically stretched. Data for WT and NM IIA-KO reproduced from (D) of Hippler et al., 2020, therefore only the mean values are shown (originally published under the Creative Commons Attribution-Non Commercial 4.0 International Public License (CC BY-NC 4.0; https://creativecommons.org/licenses/by-nc/4.0/. Further reproduction of this panel would need to comply with the terms of this license)). A mean increase of 73 nN was observed for WT cells and no force increase for NM IIA-KO cells (mean value = 0.29 nN). Compared to WT cells, NM IIB-KO cells display a significantly lower force increase (mean value = 41 nN), while NM IIC-KO cells show a comparable mean value. However, higher variations in the force response are observed for NM IIC-KO cells. Scale bar represents 10 µm in (A).

Figure 5—figure supplement 1. Initial force measurements of WT and NM II-KO cells.

To measure initial forces, cells were seeded on 3D microscaffolds without stimuli-responsive hydrogels and the displacement of the adhesive bars was tracked before and after the addition of trypsin. In all graphs, dashed lines denote reference points at the start of the measurement, at the time point of trypsin addition and at the end of the experiment. Exemplary mean displacement traces of a single WT (A), NM IIA-KO (B), NM IIB-KO (C), and NM IIC-KO (D) cell correspond to Figure 5—animations 1–4. No displacement was observed upon the addition of trypsin to NM IIA-KO cells, while WT, NM IIB-KO, and NM IIC-KO cells each showed a striking displacement. Note that the displacement in (B) at ~ 100 min resulted from focus issues in Figure 5—animation 2.

Figure 5—figure supplement 2. Displacements of individual beams for stretched WT and NM II-KO cells.

Cells were seeded on stimuli-responsive 3D microscaffolds and the displacements of individual adhesive beams during the stretch-release cycle was tracked. (A-D) The displacement traces from this figure correspond to the mean displacement traces in Figure 5A–D and Figure 5—animations 5–8. Dashed lines denote the reference point at the start of the measurement. (A) For the WT cell, all individual traces show a force increase after the stretch was applied and a force decrease after the stretch was released. (B) No force response was observed when stretching NM IIA-KO cells. (C) Individual beam displacements of a NM IIB-KO cell that show oscillatory motions in the cellular response, after the stretch was released (compare the red trace, corresponding to the right bar in Figure 5—animation 7). (D) Individual beam displacements of a NM IIC-KO, showing a force increase after the stretch but no force decrease during the time frame after the release.

Figure 5—animation 1. Initial forces of a U2OS WT cell in a 3D-printed microscaffold.

Cells were cultivated in 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 1.2 min using DIC at 40× magnification. The cell exerts force on the scaffold, thus displacing the adhesive beams. After 70 min, the imaging medium was replaced by 2 ml Trypsin/EDTA to detach the cell from the substrate and imaging was continued for another 70 min to ensure complete relaxation of the scaffold.

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Figure 5—animation 2. Initial forces of a U2OS NM IIA-KO cell in a 3D-printed microscaffold.

Cells were cultivated in 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 1.2 min using DIC at ×40 magnification. Although the cell is moving in the scaffold, no displacements of the adhesive beams were observed. After 104 min, the imaging medium was replaced by 2 ml Trypsin/EDTA to detach the cell from the substrate and imaging was continued for another 50 min to ensure complete relaxation of the scaffold.

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Figure 5—animation 3. Initial forces of a U2OS NM IIB-KO cell in a 3D-printed microscaffold.

Cells were cultivated in 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 1.2 min using DIC at ×40 magnification. The NM IIB-KO cell displaces the adhesive beams of the scaffold. After 62 min, the imaging medium was replaced by 2 ml Trypsin/EDTA to detach the cell from the substrate and imaging was continued for another 50 min to ensure complete relaxation of the scaffold.

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Figure 5—animation 4. Initial forces of a U2OS NM IIC-KO cell in a 3D-printed microscaffold.

Cells were cultivated in 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 0.3 min using DIC at 40× magnification. The NM IIC-KO cell displaces the adhesive beams of the scaffold. After 23 min, the imaging medium was replaced by 2 ml Trypsin/EDTA to detach the cell from the substrate and imaging was continued for another 13 min to ensure complete relaxation of the scaffold.

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Figure 5—animation 5. Reactive forces of a U2OS WT cell in a 3D-printed microscaffold upon a stretch-release cycle.

Cells were cultivated in stimuli-responsive 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 1.2 min using DIC at 40× magnification. After 13 min, the imaging medium was replaced by 2 ml imaging medium containing 20 mM 1-Adamantanecarboxylic acid to apply the stretch. The cell counteracts the stretch by increasing its force, thus pulling the beams inwards. After stretching the cell for 30 min, the medium was replaced by normal imaging medium to release the stretch and the cell was monitored for another 30 min, where the force again decreases, as indicated by the outward-directed beam displacement.

elife-71888-fig5-video5.gif ^{(2.8MB, gif)}

Figure 5—animation 6. Reactive forces of a U2OS NM IIA-KO cell in a 3D-printed microscaffold upon a stretch-release cycle.

Cells were cultivated in stimuli-responsive 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 1.2 min using DIC at 40× magnification. After 15 min, the imaging medium was replaced by 2 ml imaging medium containing 20 mM 1-Adamantanecarboxylic acid to apply the stretch. During the stretching time frame of 75 min, no beam displacement was observed. Similar, the cells did not displace the beams for 50 min, after the stretch was released.

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Figure 5—animation 7. Reactive forces of a U2OS NM IIB-KO cell in a 3D-printed microscaffold upon a stretch-release cycle.

Cells were cultivated in stimuli-responsive 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 1.7 min using DIC at 40× magnification. After 10 min, the imaging medium was replaced by 2 ml imaging medium containing 20 mM 1-Adamantanecarboxylic acid to apply the stretch. The cell counteracts the stretch by increasing its force, thus pulling the beams inwards. After stretching the cell for 30 min, the medium was replaced by normal imaging medium to release the stretch and the cell was monitored for another 50 min, where the force again decreases, as indicated by the outward-directed beam displacement. Note that right beam repeatedly moves outwards and back inwards, indicating oscillatory force patterns.

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Figure 5—animation 8. Reactive forces of a U2OS NM IIC-KO cell in a 3D-printed microscaffold upon a stretch-release cycle.

Cells were cultivated in stimuli-responsive 3D-printed microscaffolds, coated with 10 µg ml⁻¹ FN and imaged with a frame rate of 1 picture per 0.7 min using DIC at 40× magnification. After 20 min, the imaging medium was replaced by 2 ml imaging medium containing 20 mM 1-Adamantanecarboxylic acid to apply the stretch. The cell counteracts the stretch by increasing its force, thus pulling the beams inwards. After stretching the cell for 40 min, the medium was replaced by normal imaging medium to release the stretch and the cell was monitored for another 30 min. Besides small oscillations, no clear force decrease was observed during this time frame.

elife-71888-fig5-video8.gif ^{(12MB, gif)}