Effect of myosin II inhibition on actin polarization, centrosome positioning, and lumen initiation. (A) Effect of BB on lumen initiation. MDCK cells were seeded to grow cysts on collagen I–coated micropatterns of different sizes for 24 h. Cell cultures were treated with 50 µM BB for 30 min and then fixed and stained for F-actin, gp135, and nuclei. Cells were analyzed by confocal microscopy (z-stack projections are shown). Gray circles indicate pattern shape. An x-z view is shown for each image. Arrows indicate peripheral actin fibers. Arrowheads indicate junctional actin polarization and normal apical membrane formation. (B) Effect of BB on centrosome positioning. MDCK cells were seeded on micropatterns of different sizes, and cysts were grown for 24 h. Cell cultures were treated with 50 µM BB for 30 min and then fixed and stained for DNA, γ-tubulin, and β-catenin (red). Cells were analyzed by confocal microscopy (z-stack projections are shown). Gray circles indicate pattern shape. An x-z cross section is shown for each image. Arrowheads indicate centrosome localization. (C) Quantification of BB effect on lumen initiation. (D) Quantification of BB effect on centrosome positioning. NC, nucleus–centrosome. (E) Quantification of BB effect on internuclear distance in 1,600-µm2 micropatterns. Values are means ± SD from three independent experiments (n ≥ 30 cysts/experiment; *, P < 0.005). Bars, 10 µm.
