Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation

Abstract

The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.

FIGURE 1:

Graded control over the expression of a constitutively active form of MLCK (CA-MLCK) and ROCK2 (CA-ROCK2). (A) Schematic of doxycycline-inducible lentiviral system, where X encodes MLCK, ROCK1, or ROCK2. (B) Representative Western blot showing expression levels of endogenous MLCK, CA-MLCK, and GAPDH in U2OS (left) and U373MG cells (right) as a function of doxycycline concentration. U2OS cells were probed with rabbit ant-MLCK (Abcam 76092) and U373MG cells were probed with mouse anti-MLCK (Sigma M7905). Expression levels of CA-MLCK were quantified, normalized to GAPDH and to the highest doxycycline concentration for each cell line, and plotted below the respective Western blots (n = 4 blots for U2OS and n = 6 blots for U373MG). (C) Representative Western blot showing expression levels of endogenous ROCK2, CA-ROCK2, and GAPDH in U2OS (left) and U373MG (right) cells in the presence of various amounts of doxycycline. Expression levels of CA-ROCK2 were quantified, normalized to GAPDH and to the highest doxycycline concentration, and plotted below the respective Western blots (n = 10 blots for U2OS and n = 10 blots for U373MG at the maximum doxycycline concentration). Statistical parameters shown represent the Spearman’s rank correlation coefficient (ρ) and p value.

FIGURE 2:

Graded expression of CA-MLCK alters peripheral SF architecture, whereas CA-ROCK2 expression alters central SF architecture. F-actin images of (A) U2OS CA-MLCK and (B) U373MG CA-MLCK cultured in various doxycycline concentrations. Arrowheads point to peripheral SFs and insets highlight peripheral SFs of interest. F-actin images of (C) U2OS CA-ROCK2 and (D) U373MG CA-ROCK2 cultured in various doxycycline concentrations. Arrowheads point to central SFs and insets highlight central SFs of interest. Fluorescence intensity was normalized to the 0 ng/ml doxycycline concentration for all panels with the exception of B where the 0 ng/ml doxycycline condition is set at a higher intensity than the others. Scale bars = 10 µm; inset scale bars = 2 µm.

FIGURE 3:

Graded increases in CA-MLCK and CA-ROCK2 produce graded changes in p-RLC and pp-RLC. (A) Representative Western blots probed for p-RLC and pp-RLC in U2OS CA-MLCK (top) and U373MG CA-MLCK (bottom). Phosphorylation levels were quantified, normalized to GAPDH and CA-MLCK + 0 ng/ml doxycycline for each cell line, and plotted below the respective Western blots. p-RLC is shown by empty gray circles, whereas pp-RLC is shown by black triangles (U2OS: n = 6 blots for p-RLC [mouse] and 7 blots for pp-RLC [rabbit] blots; U373MG: n = 8 blots for p-RLC [mouse] and n = 9 blots for pp-RLC [rabbit]). (B) Representative Western blots probed for pp-RLC and p-MCL in U2OS CA-ROCK2 (top) and U373MG CA-ROCK2 (bottom). Phosphorylation levels were quantified, normalized to GAPDH and CA-ROCK2 + 0 ng/ml doxycycline for each cell line, and plotted below the respective Western blots. p-RLC is shown as empty, black circles, whereas pp-RLC is shown as solid, black triangles (U2OS: n = 4 blots for p-RLC [mouse] and n = 11 blots for pp-RLC [rabbit] expression; U373MG: n = 9 blots for pp-RLC [rabbit] and n = 6 blots for p-RLC [mouse]). Statistical parameters shown represent the Spearman’s rank correlation coefficient (ρ) and p value.

FIGURE 4:

CA-MLCK expression regulates RLC phosphorylation in peripheral SFs, whereas CA-ROCK2 expression regulates RLC phosphorylation in central SFs. Representative fluorescence images of (A) U2OS CA-MLCK (left) and U373MG CA-MLCK (right) cells and (B) U2OS CA-ROCK2 and U373MG CA-ROCK2 cells cultured in the presence and absence of doxycycline stained for p-RLC (top row, third and fourth rows in magenta) and pp-RLC (second row, third and fourth rows in green). Scale bars = 10 µm; inset scale bars = 5 µm. Fluorescence intensity of all images is normalized to that of 0 ng/ml doxycycline for each condition. (C) Quantification of immunofluorescence intensity of p-RLC (top row) and pp-RLC (bottom row) within central (gray) and peripheral (black) SFs for U2OS and U373MG CA-MLCK cells cultured in the presence and absence of doxycycline (n = 46, 46, 41, 55, 62, 53, 60, and 51 cells collected from three independent experiments for each condition, respectively, from left to right). Intensities were normalized to the average intensity value of either U2OS or U373MG CA-MLCK cells cultured in the absence of doxycycline for each experiment. (D) Quantification of immunofluorescence intensity of p-RLC (top row) and pp-RLC (bottom row) within central (gray) and peripheral (black) SFs for U2OS and U373MG CA-ROCK2 cells cultured in the presence and absence of doxycycline (n = 41, 32, 39, 31, 75, 72, 75, and 71 cells collected from three independent experiments analyzed per condition, respectively, from left to right). Intensities were normalized to the average intensity value of either U2OS or U373MG CA-ROCK2 cells cultured in the absence of doxycycline per each experiment (**p < 0.01; ***p < 0.001; Mann-Whitney test).

FIGURE 5:

CA-MLCK and CA-ROCK2 regulate the viscoelastic properties of distinct SF subpopulations. (A) SF retraction analysis. D_a: SF material destroyed by ablation; 2L: distance between SF ends over time (L is the retraction distance of a severed SF fragment); t: time. L-t curves for each stress fiber are fitted to a Kelvin-Voigt model to determine L_o, whose magnitude correlates with the SF’s dissipated elastic energy, and τ, the viscoelastic time constant, which reflects the ratio of viscosity to elasticity. (B) L_o and τ values of peripheral SF ablation for U2OS CA-ROCK2 and CA-MLCK cells cultured in the presence and absence of doxycycline (n = 21, 32 for U2OS CA-ROCK2, n = 42, 47 for U2OS CA-MLCK). (C) L_o and τ values of central SF ablation for U2OS CA-ROCK2 and CA-MLCK cells cultured in the presence and absence of doxycycline (n = 49, 51 for U2OS CA-ROCK 2, n = 22, 19 for U2OS CA-MLCK). Boxes represent 25th and 75th percentiles; whiskers represent 10th and 90th percentiles. Cross represents the mean of the distribution. Statistical differences calculated using Mann-Whitney (*p < 0.005, **p < 0.0005). Scale bars = 10 µm.

FIGURE 6:

SF mechanical properties increase in a graded manner following graded expression of RLC phosphorylation. (A) L_o and τ values of peripheral (left, dark gray) and central (right, orange) SF ablation of CA-MLCK cells cultured in various concentrations of doxycycline (n = 42, 24, 39, 37, 25, and 47 cells for peripheral SF ablation of CA-MLCK, n = 22, 14, 7, 14, 9, and 19 cells for central SF ablation). (B) L_o and τ values of peripheral (left, dark gray) and central (right, orange) SF ablation of CA-ROCK2 cells cultured in various concentrations of doxycycline (n = 21, 15, 6, 16, 6, 14, and 21 cells for peripheral SF ablation; n = 49, 41, 31, 25, 40, and 38 cells for central SF ablation). A, B, and C statistical families show statistical differences (p < 0.05) determined using Dunn’s test for multiple comparisons of nonnormally distributed data. Boxes represent 25th and 75th percentiles; whiskers represent 10th and 90th percentiles. Cross represents the distribution mean. (C) Viscoelastic retraction parameters L_o and τ of peripheral (black) and central SFs (orange) of U2OS CA-MLCK cells plotted vs. the observed increase in p-RLC (replotted from Figure 3A). (D) Viscoelastic retraction parameters L_o and τ of peripheral (black circles) and central SFs (solid orange circles) and peripheral (black circles) of U2OS CA-ROCK2 cells plotted vs. the observed increase in pp-RLC (replotted from Figure 3B). Orange values correspond to central SF ablation Spearman’s rank correlation coefficient analysis, whereas black values correspond to Spearman’s rank correlation coefficient analysis for peripheral SF ablation. Error bars of x-axis values were determined based on Western blot quantifications shown in Figure 3. All error bars represent SEM.

FIGURE 7:

Expression of phosphosmimetic p-RLC and phosphomimetic pp-RLC phenocopy the changes in SF viscoelasticity induced by CA-MLCK and CA-ROCK. Representative images of (A) U2OS RFP-LifeAct GFP-RLC AD and (B) U2OS RFP-LifeAct GFP-RLC DD. Images are taken using the GFP channel for the phosphomimetic constructs and phalloidin for SFs. White arrows point to peripheral SFs, whereas yellow arrows point to central SFs. (C) Quantification of GFP signal localization as a ratio of localization on peripheral over central SFs (n = 36 for GFP-RLC AD and 30 for GFP-RLC DD). (D) L_o and τ values of peripheral SF ablation for U2OS RFP-LifeAct, U2OS RFP-LifeAct GFP RLC-AD, and U2OS RFP-LifeAct GFP RLC-DD cells (n = 21, 28, and 28 cells, respectively). (E) L_o and τ values of central SF ablation for U2OS RFP-LifeAct, U2OS RFP-LifeAct GFP RLC-AD, and U2OS RFP-LifeAct RFP RLC-DD cells (n = 22, 23, and 31 cells, respectively). Boxes represent 25th and 75th percentiles; whiskers represent 10th and 90th percentiles. Cross represents the distribution mean. Statistical differences calculated using Mann-Whitney tests (*< 0.05, **p < 0.001, ***p < 0.0001). Scale bars = 10 µm.

FIGURE 8:

Model of subcellular regulation of RLC phosphorylation and SF viscoelastic properties. MLCK-induced p-RLC localizes and regulates the viscoelastic properties of peripheral SFs, whereas ROCK1 and 2–induced pp-RLC localizes and regulates viscoelastic properties of central SFs.