Fig. 6: Caldesmon maintains the regular distribution of myosin II along stress fibers.

a Representative examples of wild-type and oligo2 KO (indicated by dashed lines) U2OS cells mixed with each other, and stained with antibodies against Caldesmon, NM-IIA coiled-coil, and by phalloidin to visualize F-actin. Panels on the right are magnifications of the regions indicated with white boxes in the panels on the left, and they demonstrate regular patterns of NM-IIA (panel 1) along F-actin stress fibers (panel 2) in a wild-type cell. The distribution of NM-IIA filaments and filament stacks was irregular in Caldesmon knockout cells (panel 3), while the actin filaments still displayed uniform distribution also along those stress fiber regions devoid of NM-IIA (panel 4). Scale bars left 20 µm, and magnified images 5 µm. The images are representative of four experiments. b Blind analysis of the percent of wild-type and Caldesmon KO cells demonstrating regular, intermediate, and irregular distribution of NM-IIA filaments. Caldesmon depletion disturbs the normal distribution of NM-IIA filaments. The data represents wild-type (n = 193) and Caldesmon KO (n = 267) cells from four experimental repeats. The boxes indicate SD and the middle lines in the boxes are the mean values. The statistical significance was analyzed by two-way ANOVA with Tukey’s multiple comparisons test. p-values: *p = 0.0433; ***p  =  0.0001; ****p  <  0.0001. c Representative image of a Caldesmon KO cell (indicated by dashed line), and a knockout-rescue cell expressing full-length mCherry-Caldesmon. Cells were stained with an antibody against NM-IIA coiled-coil and with phalloidin. The magnified images (right) display regular NM-IIA filament distribution in the rescue cell expressing mCherry-Caldesmon (panel 1), whereas the non-transfected knockout cell (panel 2) displays irregular NM-IIA distribution along stress fibers. Scale bars, 15 and 5 µm in the whole cell and magnified images, respectively. d Representative time-lapse Zeiss Airyscan images of wild-type and Caldesmon KO cells expressing RFP-actin and GFP-NM-IIA. Magnified time frames (right) of the areas indicated by dashed boxes on left demonstrate the regular centripetal flow of NM-IIA stacks along transverse arcs in wild-type cells, whereas abnormal lateral ‘sliding’ of NM-IIA filaments occurs in Caldesmon knockout cells. This leads to NM-IIA accumulation in certain stress fiber regions (indicated by white brackets), while other stress fiber regions become largely devoid of NM-IIA. Scale bars, 5 and 1 µm in left and in magnified time-frames, respectively. Source data are provided as a Source Data file.