Figure 2.

ROCK and myosin II inhibition induce lateral lumen polarity in the absence of cell–cell adhesion. MDCK cells dislodged with Cellstripper and cultured at confluence for 16 h in SMEM in the presence of 50 μM blebbistatin, 20 μM Y-27632, or dimethyl sulfoxide (DMSO) 1:2000 (control). Where indicated (bar graph in B), cells were trypsinized and cultured on collagen IV-coated coverslips (10 μg/cm²) for 18 h. (A) Top, confocal x-y and x-z views of gp135, phalloidin, and ZO-1 in control, blebbistatin- and Y-27632–treated cells. Inset, developing lateral lumen between two cells (contours traced in white) labeled for gp135 (green) and ZO-1 (red). Bottom, merged confocal x-y views depicting Scribble, Dlg, and E-cadherin (green) in control and blebbistatin-treated cells. Inset in the scribble panel indicates the absence of scribble from the luminal domain (arrow, gp135 in red). (B) Bar graph, percentage of cells with lateral lumina treated with DMSO, blebbistatin, or Y-27632 or transfected with a control morpholino antisense oligo (contr.MA) or a myosin IIA-morpholino (myoIIA MA). Immunoblots, top, myosin IIA and actin in total lysates from cells expressing control MA (lanes 1 and 3) and myoIIA MA (lanes 2 and 4) representing two different experiments. Bottom, total cell lysates from DMSO-, ML-7–, and Y-27632–treated cells probed for T18/S19 phosphorylation of MLC, S19-MLC phosphorylation and total MLC. Bars, 10 μm.