Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins

Figures

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  FIGURE 1:. Organization of myosin II filaments in REF52 cells depleted for tropomyosin. (A) Representative images of REF52 cells transfected with nontargeting siRNA (Ctrl), siRNA against tropomyosin 1 (Tpm1), tropomyosin 2 (Tpm2), tropomyosin 3 (Tpm3), tropomyosin 4 (Tpm4), and a combination of tropmyosin 1, 2, 3, and 4 (TpmT), F-actin labeled with phalloidin and immunolabeled for myosin IIA. (B) Representative image of myosin IIA immunolabeled REF52 cells overexpressing tropomyosin 3.1 (Tpm3.1 OE). Images were taken with a SIM microscope. Scale bar is 10 µm.

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  FIGURE 2:. Analysis of myosin organization along and orthogonal to stress fibers. (A) Line scan across myosin stacks is shown in a representative image immunolabeled for myosin IIA (left). Representative profiles of line scanning for Ctrl, TpmT KD, and Tpm3.1 overexpression are presented (right). (B) Graphs of mean amplitude and peak frequency for different KD groups and Tpm3.1 overexpression. The number of line scans is n = 90 (Ctrl), n = 124 (KD Tpm3), n = 93 (KD TpmT), and n = 71 (Tpm3.1 OE). The images for analysis were taken with a W1 spinning-disk microscope. (C) Representative myosin IIA image (immunostaining) and its thresholded image to identify the length of myosin stack (left). The number of myosin stacks longer than 500 nm identified for different groups (middle). Average lengths of myosin stack per image are shown for different groups (right). The number of images is n = 18 (Ctrl), n = 11 (KD Tpm3), n = 24 (KD TpmT), and n = 10 (Tpm3.1 OE). The images for analysis were taken with a W1 spinning-disk microscope.

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  FIGURE 3:. Competition between alpha-actinin and tropomyosin for actin filaments and its effect on the organization of myosin II filaments. (A) Representative images of immunolabeled myosin IIA and alpha-actinin in Ctrl, TpmT KD, and Tpm3.1 OE cells. Scale bar is 20 µm. (B) Quantification of fluorescence intensity of myosin IIA and alpha-actinin in the stress fibers of Ctrl, TpmT KD, and Tpm3.1 OE cells. The statistical differences are shown in the graphs. The number of cells n = 12 (Ctrl), n = 9 (KD TpmT), and n = 9 (Tpm3.1 OE). (C) Representative images of immunolabeled myosin IIA and tropomyosin 3 in Ctrl and KD Actn4 cells. Scale bar is 20 µm. (D) Quantification of fluorescence intensity of tropomyosin and myosin IIA in the stress fibers of Ctrl and Actn4 KD cells. The statistical differences are shown in the graphs. The number of cells n = 16 (Ctrl), n = 9 (KD Actn4). (E) Representative image of myosin II A (RLC-GFP) and alpha-actinin-4 (alpha-actinin-4 mCherry) in cells overexpressing alpha-actinin-4. The scale bar is 5 µm. For A–D, the representative images and images for intensity analysis were acquired on a W1 spinning-disk microscope. For E, the representative images were obtained…

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  FIGURE 4:. Focal adhesion characteristics and force measurements in REF52 cells depleted for tropomyosin. (A) F-actin stained with phalloidin and focal adhesions labeled with vinculin antibody in cells transfected with nontargeting siRNA (Ctrl), tropomyosin-3 siRNA (Tpm3), and siRNA against all tropmyosins (TpmT). Scale bar is 20 µm. (B) Distribution of focal adhesions size. (C) Distribution of focal adhesion aspect ratio. Images analyzed: n = 14 (Ctrl), n = 26 (KD Tpm3), and n = 20 (KD TpmT). The images were acquired on a W1 spinning-disk microscope. (D) Representative images of myosin regulator light chain (RLC-GFP) at time point of laser ablation are shown in green for Ctrl and TpmT KD cells. Scale bar is 20 µm. Associated kymographs of F-Tractin-tdTomato (magenta) and RLC-GFP (green) along a line drawn crossing the gap are displayed in the next row. Scale bar is 5 µm. Recoil velocity and maximum opening are characterized. The differences between groups are shown in the graph. The number of images quantified for opening: n = 26 (Ctrl), n = 10 (KD Tpm3), and n = 20 (KD TpmT). The number of movies quantified for recoil velocity: n = 15 (Ctrl), n = 8 (KD Tpm3), and n = 9 (KD TpmT). The…

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  FIGURE 5:. Depletion of alpha-actinin increases disorder of myosin II filaments along with increases in focal adhesion size, actomyosin tension, and traction forces. (A) Representative images of REF52 cells transfected with nontargeting siRNA (Ctrl), α-actinin-4 siRNA (KD Actn4), and the double KD of α-actinin-1 and -4 (KD Actn1&4), and immunolabeled for myosin IIA. Scale bar is 10 µm. (B) Quantification of myosin stack length and number of stacks longer than 500 nm identified. Images analyzed: n = 18 (Ctrl), n = 28 (KD Actn4), and n = 9 (KD Actn1&4). (C) Line scanning across myosin is shown in representative images immunolabeled for myosin IIA. Representative intensity profiles for Ctrl, KD Actn4, and KD Actn1&4 are presented. Number of line scans analyzed: n = 90 (Ctrl), n = 85 (KD Actn4), and n = 89 (KD Actn1&4). (D) Representative images of actin stained with phalloidin and immunolabeled vinculin shown for Ctrl, KD Actn4, and KD Actn1&4 cells. Scale bar is 20 µm. (E) Quantification of focal adhesions in Ctrl, KD Actn4, and KD Actn1&4 cells. Images analyzed: n = 14 (Ctrl), n = 22 (KD Actn4), and n = 8 (KD Actn1&4). The images were taken with a W1 spinning-disk microscope.

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  FIGURE 6:. Depletion of alpha-actinin leads to higher tension and larger traction forces. (A) Representative images of myosin regulator light chain (RLC-GFP) at time point of laser ablation are shown in green for Ctrl and Actn1&4 KD cells. Scale bar is 20 µm. Associated kymographs of F-tractin-tdTomato (magenta) and RLC-GFP (green) along a line drawn crossing the gap are displayed in the next row. Scale bar is 5 µm. Maximum opening and recoil velocity are characterized. The differences between groups are shown in the graph. The number of images quantified for opening: n = 26 (Ctrl), n = 8 (KD Actn1&4). The number of movies quantified for recoil velocity: n = 15 (Ctrl), n = 7 (KD Actn1&4). The images were taken with a Nikon A1R confocal microscope. (B) Traction force measurements for Ctrl (n = 55) and KD Actn4 cells (n = 77). Left, representative merged images of fibronectin-Atto-647N (magenta), F-tractin-tdTomato (green), and traction force vectors for each pillar (yellow). Middle, merge and zoomed image (position indicated on left image) of RLC-GFP (green) and F-tractin-tdTomato (magenta) with a top-hat filter applied. Right, interpolated traction force map. Scale bar is 20 μm. Net…

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  FIGURE 7:. Schematic model of the reciprocal regulation of actomyosin structure and contractility by alpha-actinin-4 and tropomyosins. Tropomyosin 1-4, alpha-actinin 4, and myosin II filaments all compete for binding along F-actin. The balance between them dictates the degree of order (myosin stacks) and amount of tension generated by the actomyosin network, with alpha-actinin 4 promoting more order and less tension, whereas tropomyosin promotes less order and higher tension. The drawn lines reflect our phenotypic observations and do not imply direct interactions.