Journal: Nature Communications

Article Title: Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments

doi: 10.1038/s41467-022-33688-w

Figure Lengend Snippet: a Representative Western blot image showing Caldesmon protein levels in whole cell lysates of control and Caldesmon oligo1 and oligo2 knockout (KO) U2OS clones. The Caldesmon KO clones 2 generated with oligo1 and oligo2 Cas9 constructs (lanes 2 and 6, indicated in green) were chosen for further analysis. GAPDH was used as loading control. b Representative wide-field images of wild-type and Caldesmon oligo2 knockout U2OS cells grown on fibronectin-coated crossbow-shaped micropatterns and stained with vinculin-specific antibody and phalloidin. Caldesmon-depleted cells display less organized stress fiber networks compared to wild-type cells. Scale bars, 10 µm. c and d Analysis of cell circularity (panel c ) and cell size (panel d ) was carried out by high-content imaging of wild-type, oligo1 KO, and oligo2 KO cells, as well as oligo1 KO and oligo2 KO rescue cells expressing GFP-Caldesmon. Data represents mean ± SD n = 58,600 (wild-type), n = 11,783 (oligo1 KO), n = 13,137 (oligo2 KO), n = 61,758 (oligo1 KO-rescue) and n = 49,997 (oligo2 KO-rescue) cells analyzed from two repeats. Each data point in the graphs represents the mean value of a single well from a 96-well plate. Groups were compared with one-way ANOVA followed by Tukey’s multiple comparison test. The p -values were adjusted for multiple comparisons. The error bars indicate 0.05 significance level with 95% CI. p -values: **** p = 0.0001. e Graphical representation of random migration velocities obtained by high content imaging of wild-type ( n = 30), oligo1 KO ( n = 37), oligo2 KO ( n = 36), oligo1 KO-rescue ( n = 24) and oligo2 KO-rescue ( n = 34) U2OS cells. Data represent mean ± SD from four experiments. f Representative random migration tracks of wild-type, oligo2 KO, and oligo2 KO-rescue U2OS cells demonstrating the directionality of cells. The values on X - and Y -axis denote the extent of migration by cells from their respective mean positions. g Representative examples of a wound healing assay illustrating the scratch area covered by wild-type and oligo1 KO cells at 0, 12, and 24 h time points. Scale bars, 400 µm. h Graphical analysis of percent (%) wound area covered by wild-type, oligo1 KO and oligo2 KO U2OS cells. The X -axis represents the time point h at which the % wound area covered ( Y -axis) was calculated. The values represent mean ± SD from three independent experiments. Source data are provided as a Source Data file.

Article Snippet: For the analysis of cell shape and size, wild-type and Caldesmon KO untransfected/mCherry-Caldesmon transfected U2OS cells were plated in a fibronectin-coated 96-well plate (655180, Greiner) and incubated for 6 h. The plated cells were counter-stained with DAPI, and phalloidin 488.

Techniques: Western Blot, Knock-Out, Clone Assay, Generated, Construct, Staining, Imaging, Expressing, Migration, Wound Healing Assay

Journal: Nature Communications

Article Title: Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments

doi: 10.1038/s41467-022-33688-w

Figure Lengend Snippet: a Representative images of traction force maps of wild-type, oligo2 KO, and oligo2 KO-rescue U2OS cells grown on 26 kPa polyacrylamide covered with 488 fluorescent nanobeads and coated with collagen-1. The color scale indicates the stresses from 0 to 3000 Pascal (Pa). Scale bars, 20 µm. b Quantification of root means square (RMS) tractions of wild-type, oligo2 KO, and oligo2 KO-rescue U2OS cells. Data represents mean ± SD/SEM from n = 36 (wild-type), n = 49 (oligo2 KO) and n = 21 (oligo2 KO- rescue) cells from three experiments (one way ANOVA followed by Brown–Forsythe’s and Bartlett’s test). p -values: * p = 0.0133; **** p = 0.0001. c Representative images of GFP-Lifeact expressing wild-type and oligo2 KO U2OS cells grown on fibronectin-coated circular micropatterns. White dashed boxes in the whole cell images (left) indicate the magnified regions (right). These illustrate the retrograde flow and condensation of the transverse arc network at indicated time points. Scale bars for whole cell images and magnified regions, 10 and 2 µm, respectively. d Retrograde flow rates of transverse arcs from wild-type ( n = 32) and oligo2 KO ( n = 28) filaments from four experiments. The data represents mean ± SD (Unpaired, nonparametric, two-tailed Mann–Whitney test). e Representative examples of wild-type and oligo2 KO (indicated by dotted lines) cells, mixed with each other, and grown on 8 and 50 kPa substrates. Cells were stained with antibodies against Caldesmon and YAP, as well as with DAPI to visualize nuclei. Scale bars, 20 µm. f Graphical representation of nucleus/cytoplasmic ratios of YAP in wild-type ( n = 41) and oligo2 KO ( n = 55) cells grown on 50 kPa matrix. g Graphical representation of nucleus/cytoplasmic ratios of YAP in wild-type ( n = 47) and oligo2 KO ( n = 46) cells grown on 8 kPa matrix. Data were analyzed using an unpaired, non-parametric Mann–Whitney test with two-tailed p -values representing mean ± SD in ( f ) and ( g ). Statistical significance for d , f and g : ns ( p > 0.05; * p < 0.0332); **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: For the analysis of cell shape and size, wild-type and Caldesmon KO untransfected/mCherry-Caldesmon transfected U2OS cells were plated in a fibronectin-coated 96-well plate (655180, Greiner) and incubated for 6 h. The plated cells were counter-stained with DAPI, and phalloidin 488.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Staining