Figure 5. Contractility increase breaks actin cytoskeleton symmetry and promotes microtubule stabilization. (A) Reorganization of the actin and microtubule cytoskeletons upon serum starvation. RPE1 cells were fixed 4 h after serum withdrawal, stained with phalloidin to visualize F-actin, immunostained with antibodies against α-tubulin, and compared with serum-fed cells. Images show four examples of serum-starved and serum-fed cells. (B) Nucleus positioning upon serum starvation. RPE1 cells were fixed 4 h after serum starvation, stained with phalloidin and DAPI and compared with serum-fed cells. Images show four examples of serum-starved and serum-fed cells. White dotted lines indicate the cell symmetry axis. The top graph shows measurements of nucleus height, and the bottom graph shows measurements of nucleus position in the x–y plane in fed and starved cells at various time points after serum starvation (results of two experiments, n = 75 cells). Error bars represent standard deviation. (C) Immunostaining against phosphomyosin showed an intense staining along the noncircular actin bundle (green) in serum-starved cells. White arrowheads point at centrosomes detected with anticentrin1 antibodies (white). RPE1 cells were fixed 4 h after serum withdrawal. (D) Averaging of phosphomyosin fluorescence intensity levels (LUT fire), obtained by stacking and averaging 50 images per condition, showed that the myosin phosphorylation increased after serum starvation. (E) Identification of cold-resistant microtubules in serum-fed and serum-starved cells in the presence or absence of the myosin II ATPase inhibitor blebbistatin. RPE1 cells were fixed 2 h after serum withdrawal. Images show five examples of serum-starved cells, five examples of serum-fed cells, and five examples of serum-starved cells treated with blebbistatin for 2 h (LUT fire). The graph shows measurements of α-tubulin fluorescence intensity after cold shock (results of two independent experiments, n = 110 cells for each condition). Bars: (x and y) 10 µm; (z) 5 µm. **, P < 0.01; ****, P < 0.0001. Horizontal bars show mean values. a.u., arbitrary units.
Figure 5.

Contractility increase breaks actin cytoskeleton symmetry and promotes microtubule stabilization. (A) Reorganization of the actin and microtubule cytoskeletons upon serum starvation. RPE1 cells were fixed 4 h after serum withdrawal, stained with phalloidin to visualize F-actin, immunostained with antibodies against α-tubulin, and compared with serum-fed cells. Images show four examples of serum-starved and serum-fed cells. (B) Nucleus positioning upon serum starvation. RPE1 cells were fixed 4 h after serum starvation, stained with phalloidin and DAPI and compared with serum-fed cells. Images show four examples of serum-starved and serum-fed cells. White dotted lines indicate the cell symmetry axis. The top graph shows measurements of nucleus height, and the bottom graph shows measurements of nucleus position in the x–y plane in fed and starved cells at various time points after serum starvation (results of two experiments, n = 75 cells). Error bars represent standard deviation. (C) Immunostaining against phosphomyosin showed an intense staining along the noncircular actin bundle (green) in serum-starved cells. White arrowheads point at centrosomes detected with anticentrin1 antibodies (white). RPE1 cells were fixed 4 h after serum withdrawal. (D) Averaging of phosphomyosin fluorescence intensity levels (LUT fire), obtained by stacking and averaging 50 images per condition, showed that the myosin phosphorylation increased after serum starvation. (E) Identification of cold-resistant microtubules in serum-fed and serum-starved cells in the presence or absence of the myosin II ATPase inhibitor blebbistatin. RPE1 cells were fixed 2 h after serum withdrawal. Images show five examples of serum-starved cells, five examples of serum-fed cells, and five examples of serum-starved cells treated with blebbistatin for 2 h (LUT fire). The graph shows measurements of α-tubulin fluorescence intensity after cold shock (results of two independent experiments, n = 110 cells for each condition). Bars: (x and y) 10 µm; (z) 5 µm. **, P < 0.01; ****, P < 0.0001. Horizontal bars show mean values. a.u., arbitrary units.

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