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Supplementary Material

JCS088302 Supplementary Material

Files in this Data Supplement:

  • Supplemental Figure S1 -

    Fig. S1. Distance from the nuclear to the cellular centroids. The distance between the point of indentation (centre of the nucleus) and the cellular centroid was measured with ImageJ and determined to be 4.4±1.6 µm. This is well within the diameter of a typical cell nucleus which is ∼10-15 um. In the figure, vectors are plotted beginning at the cell nucleus (found at the origin) and ending at the cell centroid for n=23 typical cells. The red circle has a radius of 4.4 µm. The scale bar=4 µm.

  • Supplemental Figure S2 -

    Fig. S2. Correlated movement of FAs and stress fibres in response to local forces. An NIH3T3 cell expressing both actin-GFP and zyxin-mRFP (scale bar=24 µm). Kymographs were generated for three separate locations on the cell (vertical scale bar=2 µm). It can be seen in 1 that these fibres are sliding along their length (but not displacing laterally) which results in the movement of one FA into the image at ∼120 seconds (*) and another FA out of the image at ∼60 seconds (�) after force application. In 2 and 3, a correlated downward lateral displacement of F-actin filaments and FAs are observed (upward and downward arrows). In 3, the F-actin filament begins to retract downward after force application that results in the movement of an associated FA out of the kymograph.

  • Supplemental Figure S3 -

    Fig. S3. Changes in morphology and elasticity following treatment with anti-cytoskeletal drugs. Fixed NIH3T3 cells were stained for actin (green), tubulin (red), and DNA (blue) (scale bar=15 µm, applies to all). Cells were untreated (A) or treated with 10 µM nocodazole (B) or 10 µM Y27632 (C). Treatment with nocodazole specifically eliminates the microtubule network structure, while treatment with Y27632 largely eliminates stress fibres. The elasticity of similarly treated cells as measured over the nucleus are also reported; treatment with nocodazole and Y27632 reduces the Young�s modulus from ∼ 7 kPa to ∼4 kPa and ∼1 kPa respectively (values are the average ± s.e.m., n=10-12 cells; 5-6 force curves per cell).

  • Supplemental Figure S4 -

    Fig. S4. Effect of 10 µM Y27632 on the response of NIH3T3 cells to local forces. NIH3T3 cells were treated with 10 µM Y27632 and mechanically stimulated. An applied force of 20 nN caused an isotropic expansion around the nucleus within 20 seconds of force application (A, scale bar=15 µm). The images at t=20 and 240 seconds are overlays of the original cell shape (green) and the difference image (red) between the cell shape at 0 and 20 or 0 and 240 seconds. The diameter of each cell was measured before (d0) and after (d(t)) force application and the average relative expansion is reported over time in B. There is a significant expansion in response to 20 nN force (red) that is not apparent at 0 nN (blue). After removal of force the cell regains its former dimensions. Error bars are ± S.E.M.; n=4 for both 0 and 20 nN.

  • Supplemental Figure S5 -

    Fig. S5. Fluorescence intensity over time for photobleached and unbleached areas of actin stress fibres. The intensity of 10 individual points selected over bleached (grey points) and unbleached (black points) segments of stress fibres in a particular cell are plotted as a function of time. Superimposed is the average (red points) intensity of the bleached and unbleached regions of the stress fibres as a function of time. There is no significant trend of either recovery after photobleaching or bleaching in the bright segments. There is no significant difference in the means intensity values at the beginning and ending of the experiment (p>0.3 and 0.1 for bleached and unbleached data).

  • Supplemental Figure S6 -

    Fig. S6. Strain dynamics along stress fibres as a function of time and force application. As in Fig. 7 in the text, the length of a given stress fibre was normalized between -1 and +1 and we plotted the average measured strain as a function of position along a stress fibre. In this figure we plot the measured strain measured every 60 seconds for 240 seconds. Consistent with the data presented in Fig. 7, the measured strain fluctuates around a zero mean. Moreover, the fluctuations increase by ∼50% in response to a 20 nN mechanical stimulus. Importantly, there is a clear increase in strain fluctuations in the first minute of force application, which remains at the same level throughout the measurement. In this case n=370 locations on n=57 stress fibres were examined in n=13 cells.

  • Supplemental Figure S7 -

    Fig. S7. Percentage of tracked points moved over time in response to local forces. This plot contains the percentage of points for every cell at all forces that display a non-zero displacement as a function of time. The data was then normalized by subtracting the percentage of points that moved in the paraformaldehyde (PFA) control, and, as this represents any error in the measurement, the values corresponding to the PFA control are represented as the error bars on each point. At early times (<120 seconds) this is dominated by a direct displacement of stress fibres in response to the AFM cantilever. However, at later times (>120 seconds) these stress fibres stop moving and the downstream effect of mechanical stimulation begins to take over; namely, the force induced retraction and remodelling of stress fibres at distant sites from the contact point. As in Figure 2, n=10 cells for 5-20 nN, n=5 cells for 0nN; 40-70 points are tracked on each cell.