Effects of myosin II inhibition on the interactions between vinculin, paxillin, and talin. IPs were performed from lysates of untreated control MEFs (control) or MEFs treated with 20 µM blebbistatin (Blebb) followed by immunoblot analysis. (A) IP with anti-vinculin (Vcl) antibodies, immunoblot with anti-vinculin and antipaxillin (Pxn; left), or anti–talin 1 (Tln; right) antibodies. (B) IP with antipaxillin antibodies and immunoblotting with antivinculin antibodies. White lines indicate that intervening lanes have been spliced out. (C) IP with anti–talin 1 antibodies and immunoblotting with antivinculin antibodies are shown.
(A) Immunoblot analysis of lysates of untreated (control) and cells treated with 20 µM blebbistatin (Blebb) or with 20 µM blebbistatin and 100 µM Na3VO4 using antibodies specific to paxillin (Pxn), pY31 paxillin, pY397FAK, or FAK.
(D) Antipaxillin IPs from lysates of untreated control or cells treated with either 20 µM blebbistatin, 100 µM Na3VO4, and 20 µM blebbistatin or 100 µM Na3VO4 alone followed by analysis by PAGE and immunoblotting with antibodies to vinculin, paxillin, or PY epitopes. (E–I) EGFP-conjugated paxillin (Pxn-GFP wt) or paxillin bearing mutations of tyrosines 31 and 118 to phenylalanines (Pxn Y31/118F) or glutamic acids (Pxn Y31/118E) were expressed in MEFs and either treated with 20 µM blebbistatin or not.
(B) The time course of cell viability of isolated ventricular mouse myocytes in culture at 2, 24, and 48 h postplating (representative images). Supplementation with blebbistatin alone maintains cell viability to a greater extent that culture with BDM, blebbistatin and BDM in combination, or without any supplementation at both 24 and 48 h.
(C) Viability of myocytes assessed by PI staining at 6, 18, 24, and 48 h after plating. Supplementation with blebbistatin keeps a greater proportion of isolated mouse myocytes alive throughout the 48 h post isolation. *versus control P < 0.05.
A: cultures were incubated with 100 nM KT5926 (KT) for 60 min, and RLC phosphorylation was analyzed by glycerol/urea gel electrophoresis. Inhibition of MLCK caused a significant reduction in constitutive RLC phosphorylation. Monolayers preincubated with 100 nM KT5926 for 30 min and then treated with blebbistatin for an additional 30 min in the presence of inhibitor showed that MLCK was responsible for blebbistatin-induced RLC phosphorylation.
B: effect of blebbistatin on MLCK catalytic activity. Both MLCK210 (lane 1) and MLCK155 (lane 4) catalyze RLC phosphorylation in the presence of Ca2+/CaM, whereas in the absence of Ca2+/CaM, no 32PO4 was incorporated into myosin II RLCs (lanes 2 and 5). Blebbistatin does not directly activate MLCK since incubation of either isoform of MLCK in phosphorylation buffer containing 50 μM blebbistatin in the absence of Ca2+/CaM resulted in no RLC phosphorylation (lanes 3 and 6). Blebbistatin does not inhibit MLCK activity in the presence of Ca2+/CaM (lanes 7 and 8). Lane 1, MLCK210 + Ca2+/CaM; lane 2, MLCK210 + EGTA; lane 3, MLCK210 + EGTA + 50 μM blebbistatin; lane 4, MLCK155 + Ca2+/CaM; lane 5, MLCK155 + EGTA; lane 6, MLCK155 + EGTA + 50 μM blebbistatin; lane 7, MLCK210 + Ca2+/CaM + 50 μM blebbistatin; lane 8, MLCK155 + Ca2+/CaM + 50 μM blebbistatin.
C: effect of Ca2+ depletion on blebbistatin-induced RLC phosphorylation. For Ca2+ chelation, monolayers were preincubated for 1 h at room temperature in Ca2+-free HBSS containing 10 mM BAPTA-AM/1 μM thapsigargin. Cells were treated with blebbistatin for 30 min; samples were separated by glycerol/urea gel electrophoresis, transferred to nitrocellulose, and probed with anti-myosin RLC antibody. Ca2+ depletion prevents blebbistatin-induced RLC phosphorylation.
Palladin knockdown cells show more efficient recovery from Blebbistatin treatment. (A) DIC image of a palladin KD (Palld4) cell on a gel of intermediate stiffness (10–30 kPa range). Scale bar: 10 μm. (B) DIC image of the same cell as in A, 30 minutes after incubation in 15 μM blebbistatin. (C) DIC image of the cell 1 hour after washout from blebbistatin, showing recovery of cell morphology. (D) Traction force map of the cell in A showing robust generation of traction forces. (E) Traction force map of the cell in B, showing disappearance of traction forces upon blebbistatin addition. (F) Traction force map of the cell in C, showing recovery of traction forces 1 hour after Blebbistatin washout.
The effect of Blebbistatin on GCs morphology. (a–b) Lamellipodium emerging from a DRG neuron in control condition and after treatment with 30 μM Blebbistatin, respectively. Note the 'filopodish' appearance of the lamellipodia after Blebbistatin treatment. (c) Immunostaining of DRG lamellipodium in control condition for actin (green) and tubulin (blue) staining. (d) As in (c) but in the presence of 30 μM Blebbistatin.
(e) Immunostaining of a GC after Blebbistatin treatment for actin, NMIIA and tubulin and merge of the three staining. Arrows and arrowheads indicate filopodia with and without a clear staining for tubulin, respectively. (f) Immunostaining of a GC after Blebbistatin treatment for actin, NMIIB and tubulin and merge of the three staining.
(B) Immunofluorescence of VE-cadherin (red) and phalloidin staining of F-actin (green) following 30-min treatment with control inactive (+)-blebbistatin (left) or active (−)-blebbistatin (right) reveal reduced size of VE-cadherin puncta following myosin II inhibition. Insets are magnified with equal ratios relative to boxed regions in original image.
Rho kinase–mediated myosin II activity and substrate stiffness slow MEF migration and increase adhesion size.(C) Immunolocalization of PY epitopes (P-Tyr) to visualize adhesions (green) and fluorescent phalloidin staining to visualize actin filaments (red) under the treatments as untreated cells (control), cells treated with 20 µM blebbistatin (Blebb) or 10 µM Y27632, or plated on 1.0 kPa compliant polyacrylamide substrates. Merged images are shown in the third column, and boxed regions are magnified in the fourth column. Bars: (third column) 10 µm; (fourth column) 2 µm.
(A) Immunolocalization of paxillin (Pxn; red) and PY epitopes (P-Tyr; green) in untreated cells (control) or cells treated with 20 µM blebbistatin (Blebb). Merged images are shown in the third column, and boxed regions are magnified in the fourth column. Bars: (third column) 10 µm; (fourth column) 2 µm.
(B) Immunolocalization of paxillin (red) with either (in green); talin 1 (Tln), FAK, β1 integrin (β1-Int), zyxin (Zyx), vinculin (Vcl), or α-actinin (Actn) in untreated control cells (left) or cells treated with 20 µM blebbistatin (right). Merged images are shown in the right column. Bar, 2 µm.