Cell confinement regulates nuclear–centrosomal axis orientation after the first cell division. (A) Life-actin-GFP localization during cyst formation in different confinement conditions. Life-actin-GFP MDCK cells were seeded on collagen I micropatterns of different sizes (700 and 1,600 µm2) to grow cysts and analyzed by video microscopy for 48 h (one frame = 10 min). After 48 h, DNA was stained with cell-permeable Hoechst to show nuclei. Cysts contained an aggregate of approximately four cells, of which three nuclei are visible in the same plane. Still images were selected at different time points, and in some cases, intensity was enhanced to facilitate visualization of cell structures. Arrows indicate accumulation of actin at the cell–cell junctions or peripheral actin fibers. Arrowheads indicate lumen. Bars, 20 µm. (B) Quantification of polarization of actin during micropatterned cyst formation in collagen. Life-actin-GFP signal was analyzed after first cell division using live-cell imaging. The ratio between Life-actin fluorescent signal at junctions and periphery was quantified (n = 6; *, P < 0.05). (C) Quantification of lumen initiation in collagen I micropatterned cysts at 24 h. n ≥ 50 cysts/experiment; *, P < 0.005; **, P < 0.001. (D) Centrosome orientation in micropatterned MDCK cells. MDCK cells were seeded to grow cysts on collagen I–coated micropatterns of different sizes and fixed after 20 h. Cells were stained for F-actin, γ-tubulin, and nuclei. Cells were analyzed by confocal microscopy (z-stack projections and x-z cross sections are shown). Gray circles indicate micropattern shape. Arrowheads indicate position of the centrosome. Bars, 10 µm. (E) Scheme of procedures for quantification of centrosome and nuclear positioning in cell doublets. Distances between nuclei are quantified (d). The orientation of the nucleus–centrosome (NC) axis is considered incorrect (misoriented) when the centrosome is facing the periphery and correct (junction oriented) when the centrosome is within the 90° quadrant formed between the nucleus and the cell–cell junctions. MTOC, microtubule-organizing center. (F) Quantification of centrosome positioning in different confinement conditions. n ≥ 30 cysts/experiment; *, P < 0.005. (G) Quantification of internuclear distance. n ≥ 30 cysts/experiment; *, P < 0.005. Values are means ± SD from three independent experiments.
