Cell shape regulates cell polarity and ciliogenesis. The experimental procedure is described in Fig. 2 C. RPE1 cells were plated on small (750 µm2) and large (3,000 µm2) micropatterns and serum starved for 24 h. (A) Micropatterned cells were fixed and immunolabeled to reveal primary cilium, centrosome/basal body, actin filaments, and nucleus (Fig. S3 A). Z stacks were performed to measure centrosome position. The ratio between centrosome position and local cell height was measured in confined and extended cells before and 24 h after serum starvation in ciliated and nonciliated cells. (B) Micropatterned cells were fixed in cold methanol and immunolabeled for γ-tubulin to reveal the centrosome (green) and immunolabeled with α-tubulin to reveal microtubules (red). DNA is in blue. Z stacks were acquired and deconvolved to detect centrosome positioning (Fig. S3 B). XZ optical sections illustrate centrosome positioning above the nucleus in confined cells (left) and below the nucleus in spread cells (right). (C) Micropatterned cells were fixed with paraformaldehyde, immunolabeled for acetylated tubulin (green), and stained with phalloidin (red; Fig. S3 C). DNA is in blue. XZ optical sections illustrate the presence of the primary cilium at the dorsal surface of confined cells (left) and the presence of acetylated microtubules in the cytoplasm of spread cells (right). (D) Results summary. When cells are spatially confined, they develop a polarized actin architecture with contractile bundles in the ventral surface and a polymerizing network in membrane protrusions at the dorsal surface. In these cells, the nucleus–centrosome axis is reproducibly oriented toward the dorsal surface in a Rho kinase–dependent manner. The apical positioning of the centrosome and its anchoring in the ezrin-rich actin network promote the formation of the primary cilium. When cells are highly extended, the actin network is unbalanced toward the formation of numerous and large contractile bundles. The internal polarity is reversed compared with confined cells. The nucleus–centrosome axis is oriented toward the ventral surface in an actin- and microtubule-dependent manner. The centrosome is in close proximity to actin stress fibers, whose contractility prevents the extension of the primary cilium. X bars, 5 µm. Z bars, 2 µm.
