Nuclear–centrosomal orientation during lumen initiation requires aPKC. (A) Localization of aPKC in control or BB-treated MDCK cells. MDCK cells were seeded on micropatterns and treated with BB. Cells were fixed and stained to detect aPKC, tubulin, nuclei (blue), and γ-tubulin (γ-tub; gray). Arrowheads indicate aPKC localization at lateral plasma membrane. Arrows indicate centrosome localization. Dotted boxes show areas of magnification. (B) Quantification of lumen initiation in aPKC-PS inhibitor-treated cells. n ≥ 50 cysts/experiment. (C) Effect of aPKC inhibition in BB (Bleb)-induced lumen morphogenesis on low confinement. MDCK cells were seeded on 1,600-µm2 collagen I micropatterns and treated with 40 µg/ml aPKC-PS overnight. After 24 h, cells were treated with BB for 45 min and then were fixed and stained to detect gp135, F-actin, and tubulin. Cells were analyzed by confocal microscopy (z-stack projections and x-z cross sections are shown). Arrowheads show apical membrane. (D) Effect of aPKC inhibition on centrosome positioning. Cells grown as in C were fixed and stained to detect γ-tubulin, β-catenin, and DNA. Cells were analyzed by confocal microscopy (z-stack projections and x-z cross sections are shown). Gray circles indicate pattern shape. Arrowheads show centrosome position. (E) Quantification of centrosome positioning in aPKC inhibitor–treated cells. n ≥ 30 cysts/experiment. NC, nucleus–centrosome. (F) Quantification of internuclear distance in aPKC inhibitor–treated cells. n ≥ 30 cysts/experiment. Values are means ± SD from three independent experiments. *, P < 0.05. N, nuclei. Bars, 10 µm.
