Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices

Abstract

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied by local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micrometre elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ∼2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing.

Figure 1

Contractile Units (CUs) at cell edges require myosin. (a) Cartoon illustration of a CU at the cell edge. (b) Left: Actual CUs observed at the edge of a cell spreading on 8.4 pN/nm pillars (Experiment was repeated 7 times, 45 videos taken altogether). Arrows represent pillar movement vectors: red, contractile pairs; yellow, non-paired pillars. Cell edge is marked in blue. Right: Typical displacement vs. time of two 0.5 μm diameter pillars that were part of a CU. Experiment was repeated (c) α-actinin localizes to the cell edge during P2 stage of spreading (~15 minutes after initial attachment) but is distributed evenly during P1 (from initial attachment up to ~15 minutes). Experiment was repeated three times (10 videos altogether). (d) GFP-myosin-IIA as well as immunolabelled myosin-IIA localize to the cell edge. Experiment was repeated twice. (e) Myosin-IIA is required for force production in CUs. Typical forces generated by myosin-IIA-KD cells show a significant reduction of the inward-directed forces (see also Supplementary Fig. 1b); only ~25% of the pillars show inward movements compared to >80% in WT cells. CUs are rarely observed, and even in such cases they are short lived and cause small pillar displacements (average maximum displacement = 23±2 nm). Experiment was repeated twice (9 videos altogether). (f) Treatment of the cells with blebbistatin (50 μM) leads to a rapid halt in pillar displacement. Experiment was repeated twice (6 videos altogether).

Figure 2

Distribution of sarcomeric proteins in CUs. (a) Top: Patches of p-MLC localize between pillars at the cell edge, whereas α-actinin is localized around the pillars. Middle: Tpm overlaps with α-actinin at the edges of the pillars (arrow in zoom-in image) and is also located between pillars (arrowhead). Experiment was repeated 3 times. Bottom: Normalized average fluorescence intensities of α-actinin, p-MLC, and Tpm on 0.5 μm pillars measured from line-scans between two adjacent pillars. (n = 20 traces from 4 cells in each case). (b) Localization of sarcomeric proteins with respect to nascent adhesions in cells plated on 2D surfaces. Cells transfected with GFP-β3-integrin (marker for nascent adhesions) were fixed after 15 minutes of spreading on fibronectin-coated coverslips. p-MLC and Tpm (both imaged after immunostaining), localized between nascent adhesions (with some overlap of Tpm with the adhesions); mCherry-α-actinin co-localized with β3-integrin and also extended out of the adhesions. Experiment was repeated twice.

Figure 3

Myosin mini-filaments appear in CUs. (a) 3B super-resolution image of p-MLC (red) and α-actinin (green) in a contractile pair where the displacement of the pillars was tracked (right traces) and the final displacement vectors (about 35 nm) are marked by the arrows. Note the dumbbell shape of p-MLC, consistent with the known shape of myosin mini-filaments. Experiment was repeated 3 times (7 videos altogether). (b) Left: additional examples of dumbbell-shaped p-MLC filaments from super-resolution fluorescence analyses. Right: The length histogram of these dumbbell shapes matches the known size of myosin mini-filaments. n = 30 patches from 5 cells. (c) Schematic of a CU with the relevant molecular components.

Figure 4

Cells pull on pillars with nanometre-level steps. (a) Top: Median-averaged tracking data of a single pillar displaced by a piezo-device with 1.2 nm steps at 2 steps/s (blue), along with fitting data using the step-detection algorithm (black). Bottom: the frequency of detection and average step sizes detected (±SEM) using the step-fitting algorithm on data obtained by piezo-controlled movements with 0.6 and 1.2 nm steps at different rates. n=102,96,99 steps from 12,10,11 pillars for the 0.6 nm steps at 2,3,4 steps per second data, respectively; the mean detected step sizes were insignificantly different from 0.6 nm: p-values=0.11, 0.57, and 0.22, one-sample t-test. n=90,92,94 steps from 10,11,11 pillars for the 1.2 nm steps at 2,3,4 steps per second data, respectively; the mean detected step sizes were insignificantly different from 1.2 nm: p-values=0.19,0.27.0.3, one-sample t-test. All piezo-driven experiments were repeated 4 times for each case. (b) Top: Median-averaged displacement data of a single pillar which was part of a CU at the cell edge (green) with the raw 100 Hz measurements in grey, along with fitting data using the step-detection algorithm in black. Bottom: Negative control data along with its step-fitting data for the same pillar (grey and black, respectively). (c) Histograms of the steps detected in the real and negative control data (n=527 steps from 24 pillars in 7 cells; experiment was repeated 4 times).

Figure 5

Steps of paired pillars are simultaneous and antiparallel. (a) Image of paired pillars that began to move at the same time (orange and green arrows in image on left; cell edge shown in blue) show simultaneous anti-parallel steps during early phases of displacement. Black arrows represent the movement vectors of unpaired pillars that were being displaced at the same time (see displacement curves on the right) when the simultaneous steps of the pillar pair were detected; these were used as controls to verify that the correlated steps did not arise from similar fits of neighbouring pillar movements. (b) Histogram of the time difference between simultaneous steps in paired pillars has a peak around 0, whereas for unpaired pillars in the same field it is random. In paired pillars ~70% of the steps were correlated within the first 5 seconds of displacement compared to ~20% in unpaired pillars. (n=144 and 152 steps from 17 paired and non-paired pillars, respectively). (c) Histograms of the sum of the displacements for simultaneous steps from paired pillars on stiff (8.4 pN/nm) and soft (1.6 pN/nm) pillars. (n=70 and 74 steps from 8 and 9 CUs for the stiff and soft pillars, respectively; experiment was repeated three times).

Figure 6

Adhesion reinforcement at a specific force level is critical for rigidity sensing. (a) Typical early displacement traces showing first force production cycles on stiff and soft pillars, along with plots of the mean±SEM number of steps during such cycles (n=12,14 pillars from 4,5 cells, respectively; experiment was repeated three times). (b) Adhesion breakage observed on 0.85 pN/nm pillars (See also Supplementary Movie 3). Cell edge is marked in blue; yellow vector shows the noise level of pillar movements. Notice that the pillar marked with a red vector is released by the cell and returns to zero force position at 180 s. Experiment was repeated three times (9 videos altogether). (c) Histogram of the time interval between the peak in GFP-α-actinin intensity and the peak in pillar displacement (Δt_α, see inset time traces) shows that α-actinin recruitment precedes the peak in force production by 7.5±13 s (mean±SD; n = 20 pillars from 3 cells). Experiment was repeated twice. (d) Typical time traces of pillar displacement (stiffness: 1.6 pN/nm) and GFP-α-actinin intensity show a considerable increase in α-actinin recruitment during the pause in displacement, followed by subsequent force production. Experiment was repeated twice (8 videos altogether). ***p-value < 0.001, two-tailed, equal variance t-test.

Figure 7

Tpm2.1 regulates force production, step size, and adhesion growth. (a) Average area ±SEM of cells transfected with non-targeting SiRNA (control) and Tpm2.1-KD cells after 1 hour of spreading on stiff and soft pillars (n=50,52,51,54 cells). Experiment was repeated three times. (b) Mean±SEM of the maximal displacements of pillars when control and Tpm2.1-KD cells were plated on stiff and soft pillars (n=81,79,70,71 pillars from at least 5 cells in each case). Experiment was repeated twice. (c) Left: Typical pillar displacement by a Tpm2.1-KD cell, showing high displacement rate and large steps. Right: quantification of pillar displacement rates (mean±SEM) by Tpm2.1-KD and control cells (n=20 pillars from 3 cells in each case). Experiment was repeated three times. (d) Average step sizes for control and Tpm2.1-KD cells. Red lines are the median values, the edges of the blue boxes are the 25^th and 75^th percentiles, the whiskers extend to the most extreme data points not considered outliers, and outliers (data greater than 3 SD than the median) are plotted individually as red dots; comparison between boxes is done by the overlap of the notches: if they do not overlap, the conclusion with 95% confidence is that the true medians do differ. (n=344 and 316 steps from 20 and 15 pillars for control and KD cells, respectively). Experiment was repeated three times. (e) Adhesions are much smaller after Tpm2.1-KD. Left: Micrographs showing the distribution of paxillin-GFP in Tpm2.1-KD and control cells. Right: Quantification of adhesion sizes in Tpm2.1-KD and control cells (n=245 and 256 adhesions from 6 cells in each case). ***p-value < 0.001, two-tailed, equal variance t-test. Experiment was repeated twice.

Figure 8

Tpm2.1 differentiates between normal and malignant cell lines and controls growth on soft matrices. (a) Examples of MCF-10A and MDA-MB-231 cells plated on pillars and immunostained for Tpm. Experiment was repeated twice. (b) Average step sizes for MCF-10A cells (M-10A), MDA-MB-231 cells (M-231), MCF-7 cells (M-7), Tpm2.1-KD MCF-10A cells, and MDA-MB-231 cells with YFP-Tpm2.1 expressed. (n=231,248,245,311,270 steps from at least 12 pillars in each case). Experiment was repeated twice for each case. Red lines are the median values, the edges of the blue boxes are the 25^th and 75^th percentiles, the whiskers extend to the most extreme data points not considered outliers, and outliers (data greater than 3 SD than the median) are plotted individually as red dots; comparison between boxes is done by the overlap of the notches: if they do not overlap, the conclusion with 95% confidence is that the true medians do differ. (c) Adhesion sizes are larger in MDA-MB-231 cells when Tpm2.1 is expressed. Left: Micrographs showing the distribution of paxillin-GFP in MDA-MB-231 and MDA-MB-231+Tpm2.1 cells. Right: Quantification of adhesion sizes in Tpm2.1-KD and control cells (n=220 and 217 adhesions from 6 cells in each case). Experiment was repeated twice. (d) Soft agar assay showing growth of Tpm2.1-KD MCF-10A cells but not of control cells. Cells were stained with Crystal Violet. Experiment was repeated twice.