Figure 2. Differential regulation of myosin IIA and myosin IIB localization at apical junctions.

(a–c) Impact of Rho and ROCK on junctional localization of myosin II isoforms. MCF-7 monolayers were incubated with Y-27632 (5–10 µM), the Rho inhibitor C3-transferase (C3-T, 0.5 µg ml⁻¹) or dimethylsulphoxide (DMSO) for 3 h, then fixed and immunostained for E-cadherin and myosin IIA or myosin IIB. (a) Representative apical confocal images. (b, c) Junctional localization of myosin IIA or myosin IIB was quantified by fluorescence intensity analysis of contacts in cells treated with C3-T (b) or Y-27632 (c). Data are means and s.e.m. (n = 24). (d) MLCK supports junctional localization of myosin IIA but not that of myosin IIB. Myosin isoform localization at junctions was quantified in cells treated with ML7 (10 µM, 3 h). Data are represented as means ± s.e.m. (n = 24). (e) Myosin activity is necessary for junctional localization of myosin IIA but not that of myosin IIB. Junctional localization of myosin II isoforms was measured in cells treated with blebbistatin (blebbi.) (100 µM, 3 h). Data are represented as means ± s.e.m. (n = 24). (f, g) Rap1 supports junctional localization of myosin IIB. MCF-7 cells were transfected with siRNA against Rap 1A (100 nM) or scrambled siRNA (100 nM). (f) After 48 h cells were fixed and immunostained for Rap 1 and either myosin IIA or myosin IIB. Transfected cells that show reduced levels of Rap1 are identified with asterisks in the myosin staining. (g) Junctional localization of myosin isoforms was measured by fluorescence intensity analysis. Data are represented as means ± s.e.m. (n = 21). For all experiments: two asterisks, P < 0.01; three asterisks, P < 0.001; Student’s t-test. Scale bars, 10 µm.