Effect of myosin activation on lumen initiation. (A) Effect of myosin RLC2 mutants (RLC2-DD and RLC2-AA) on lumen initiation. MDCK cells transfected with GFP-RLC2-DD, GFP-RLC2-AA, or GFP (control) were seeded on micropatterns of different sizes, and cysts were grown for 24 h. Cell cultures were fixed and stained for gp135 and β-catenin (β-cat). 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 localization of RLC2-DD in peripheral contractile bundles. Filled arrowheads indicate apical membrane localization. Empty arrowheads indicate cell–cell junction position. Bars: (main images) 10 µm; (cross sections) 5 µm. (B) Quantification of RLC2-DD and RLC2-AA effects on lumen initiation. n ≥ 30 cysts/experiment. (C) Quantification of RLC2-DD effect on centrosome positioning in high confinement (700 µm2). n ≥ 30 cysts/experiment. NC, nucleus–centrosome. (D) Quantification of RLC2-DD effect on internuclear distance in high confinement (700 µm2). n ≥ 15 cysts. Values are means ± SD from three independent experiments. *, P < 0.05.
