Figure 5. Myosin IIB regulates the apical F-actin ring.

(a) Representative confocal images of the perijunctional F-actin ring in control and myosin II isoform KD cells identified by phalloidin staining. Detailed views of F-actin at cell–cell contacts are shown below in magnifications. Scale bars, 10 µm. (b–c) F-actin content in the perijunctional rings was quantified by line scan profile analysis of fluorescence intensity at contacts between control and myosin isoform KD cells. (b) Nonlinear fit curves of pooled intensity profiles (n = 24). Intensity profiles (means and s.e.m.) are shown in Supplementary Information, Fig. S6a. (c) Average peak fluorescence intensity values of actin in knockdown and control cells. Data are means and s.e.m. (n = 24; three asterisks, P < 0.001; Student’s t-test). (d) Junctional actin turnover in control or myosin isoform KD MCF-7 cells. Transiently transfected GFP–actin was revealed by live-cell imaging, and GFP–actin in the apical actin ring was bleached in a region of interest. FRAP recovery is represented by curve fits of GFP–actin fluorescence (n = 8); average fluorescence intensity profiles (± s.e.m.) are shown in Supplementary Information, Fig. S6b. (e) Lateral movement of the apical actin ring. Kymographs from photobleaching data were performed by using line scans perpendicular to the GFP–actin ring. Representative kymographs, an illustration of the quantification method, and scatter plots of lateral movement are shown (n = 8; two asterisks, P < 0.001; analysis of variance with Dunnett’s test). Lateral movement of the ring away from the initial position (red line) was calculated by measuring the angle between the initial position and the final position in either direction (blue dotted lines show the extremes of angles).