Myosin II governs intracellular pressure and traction by distinct tropomyosin-dependent mechanisms

FIGURE 1:. 2D matrix rigidity reciprocally regulates pressure and adhesion size. (A) Primary human dermal fibroblasts were plated on relatively soft or stiff 2D surfaces and transiently transfected with YFP-paxillin to visualize focal adhesions. Fibroblasts form larger focal adhesions on stiff (Young’s modulus 71 kPa) compared with soft (5 kPa) 2D surfaces, quantified in B (n ≥ 630, N = 3). Bars, 10 µm. *, P < 0.0001. (C) In contrast, intracellular cytoplasmic pressure (Pic) significantly increases on soft 2D surfaces (n ≥ 25, N = 3). *, P < 0.02.

FIGURE 2:. Tpm 1.6 expression in HFFs increases intracellular pressure and decreases traction stress. (A) Expression of tGFP-Tpm 1.6 in HFFs on 2D glass surfaces causes a significant elevation of intracellular pressure compared with GFP expressing control cells (n ≥ 15, N = 3). *, P < 0.0001. (B) The increase in pressure due to tGFP-Tpm 1.6 expression is reduced by treatment with the myosin II inhibitor blebbistatin (n = 18, N = 3). *, P < 0.03. (C) Measuring the cell (outlined in green) -dependent displacement (red arrows) of flexible microposts of known elasticity and position allows traction stress to be calculated. (D) Expression of both GFP-Tpm 1.6 and CFP-Tpm 2.1 decrease the traction stress produced by HFFs on the flexible microposts, despite the Tpm-1.6–dependent increase in pressure measured in A (n ≥ 18, N = 3). *, P < 0.02.

FIGURE 3:. Tpm 2.1 is not required for generating intracellular pressure but is critical for governing traction stress. (A) Representative Western blots of specific tropomyosin isoforms knockdown probed with antibodies to TPM1 (α/9d mAB), Tm1 (CG1 mAB), MLC2, NMIIA, and GAPDH. (B) Quantification of specific Tpm 1.6/7- and Tpm 2.1-siRNA show reduction of specific tropomyosin isoform level with corresponding knockdown (N = 6). *, P < 0.001. (C) Quantification of MLC2 and NMIIA shows that only Tpm 1.6/7 siRNA results in reduction of MLC2 but not NMIIA (N = 6). *, P < 0.001. (D) Specific Tpm 2.1 siRNA results in an increase in traction force compared with control siRNA-treated cells (n ≥ 15, N = 3). *, P < 0.005. (E) The treatment of HFFs plated on 2D glass with either Tpm 1.6/7 or Tpm 2.1 siRNA did not affect the generation of intracellular pressure compared with control cells (n = 17, N = 3). P > 0.5.

FIGURE 4:. Tpm 1.6/7 and Tpm 2.1 mediate their positive and negative regulation of pressure and traction force when associated with F-actin. (A) Representative Western blots of soluble and insoluble fractions from HFFs cultured on rigid, plastic tissue culture dishes for 24 h followed with 20 µM latrunculin-A (Lat-A) treatment for 1 h before lysing. Blots were probed with antibodies to β-actin, TPM1/2 (TM311 mAB), and GAPDH. (B) Quantifications represent the ratio of the insoluble to soluble fractions of β-actin, GAPDH, and tropomyosin isoforms in control compared with Lat-A–treated cells (N = 6). ***, P < 0.001; *, P < 0.05; **, P < 0.01. (C) Representative Western blots of the soluble and insoluble fractions from HFFs cultured on 0.5 and 64 kPa 2D substrates. Blots were probed with antibodies to myosin light-chain 2 (MLC2), nonmuscle myosin heavy-chain II A (NMIIA), along with the β-actin, TPM1/2, and GAPDH antibodies described in A. (D, E) Quantifications represent relative change in the insoluble protein levels of the tropomyosin isoforms, β-actin, MLC2, and NMIIA; all are normalized to β-actin (N = 6). *, P < 0.01.

FIGURE 5:. Tpm 1.6 and 2.1 are cobundled in ventral actin stress fibers. (A) The F-actin stress fiber networks within HFFs consist of ventral stress fibers, transverse arcs, and dorsal stress fibers (Tojkander et al., 2011). (B) HFF transfected with GFP-Tpm1.6 shows Tpm1.6 filaments (green) bound to F-actin (magenta) and highlights their association with dorsal and ventral stress fibers. Similarly, CFP-Tpm2.1 is found predominantly in dorsal and ventral stress fibers (n = 30, N = 3). Scale bars, 10 µm. (C) Primary human fibroblasts (HFFs) were costained with rhodamine phalloidin and Acti-stain 488 phalloidin as a positive colocalization control and imaged by TIRF microscopy. (D) TIRF images of HFFs cotransfected with GFP-Tpm1.6 and mRFP-Tpm2.1. Scale bars, 5 µm. (E) Cophalloidin-labeled cell shows both red and green phalloidin intensities are more closely correlated to each other than (F) cotransfected GFP-Tpm1.6 and mRFP-Tpm2.1 as shown in representative plot profiles. (G) Pearson’s correlation coefficient shows that red and green phalloidin staining on ventral stress fibers are more colocalized on the same actin bundle than GFP-Tpm1.6 and mRFP-Tpm2.1 in three independent experiments.…

FIGURE 6:. Migration in soft material requires Tpm 2.1. (A) Morphology of control primary human foreskin fibroblast (HFF) migrating on glass. Bar, 10 µm. (B) Velocity of Tpm 1.6/7 siRNA HFFs migrating on glass is significantly decreased compared with Tpm 2.1 and control siRNA cells (n = 36, N = 3). *, P < 0.05 vs. control cells. (C) Representative images of YAP distribution in Tpm-KD HFFs; cells were cultured on glass for 24 h before fixing and immunostaining. Bars, 10 µm. (D) Quantification of nuclear to cytoplasmic ratio of YAP in maximum projection shows a decrease in nuclear YAP in Tpm 1.6/7 siRNA or when myosin II activity is inhibited (n = 30, N = 3). *, P < 0.001 vs. control cells. (E) Morphology of control HFF in 1.7 mg/ml rat-tail collagen. Bar, 10 µm. (F) Decreased Tpm 2.1 expression decreases velocity in cells migrating in collagen compared with control cells (n = 45, N = 3). *, P < 0.05 vs. control cells. (G) Specific Tpm-KD HFFs were cultured on 0.5 kPa (soft) of stiffness for 24 h before fixing and immunostaining; shown are representative maximum projections of confocal z-stack immunofluorescence images of YAP. Bars, 10 µm. (H) Quantification of the nuclear to cytoplasmic…

FIGURE 7:. Tpm 1.6/7 is required for the nuclear piston mechanism of 3D migration and the generation of high-pressure lobopodial protrusions. (A) Representative phase image of primary human dermal fibroblast migrating in 3D CDM. Bar, 10 µm. (B) Velocity assessment of specific tropomyosin knockdown cells show Tpm1.6/7- and Tpm 2.1-siRNA–treated cells are unable to migrate efficiently in CDM compared with control cells (n = 54, N = 3). *, P < 0.001. (C) Tpm 1.6/7 knockdown results in an increase of lamellipodia (yellow arrowheads) indicated by an enrichment of cortactin (green) and F-actin (magenta) in leading protrusions. Bars, 10 µm. (D) Morphometric analysis of lamellipodia formed in specific tropomyosin knockdown cells (n ≥ 23, N = 4). *, P < 0.001. (E) Knockdown of Tpm 1.6/7 significantly decreases anterior intracellular pressure in cells in 3D CDM (n ≥ 35, N = 3). *, P < 0.005. (F) Representative images of 2D and 3D fibroblasts on and in 3D CDM transfected with GFP-Tpm 1.6, respectively. Bars, 10 µm. (G) Quantification of Tpm 1.6/7 fluorescence density in both 2D and 3D cells shows a significant increase in Tpm 1.6/7 expression in front of the nucleus in 3D lobopodial cells (n ≥ 15, N =…

FIGURE 8:. Model of the positive regulation of intracellular pressure by Tpm 1.6/7 and the negative regulation of traction force by Tpm 2.1. Mechanism 1: When fibroblasts encounter a 2D surface of increased rigidity, we propose that Tpm 2.1 (magenta) dissociates from actomyosin filaments leading to increased myosin II activity and traction force generation for efficient migration. Mechanism 2: When fibroblasts move on softer surfaces or through 3D CDM, cells have significantly higher intracellular pressure. Under these conditions, Tpm 1.6/7 (blue) is recruited to actin filaments to increase actomyosin contractility and generate intracellular pressure.