Actin network assembly, contractility, and ciliogenesis. The experimental procedure for A and B is described in Fig. 2 C. RPE1 cells were plated on micropatterns (from 750 to 3,000 µm2) and serum starved for 24 h. (A) Micropatterned cells were treated with cytochalasin D during starvation. Cells were fixed and stained for acetylated tubulin (red), F-actin (green), and DNA (blue). Primary cilium occurrence (top, n = 82, 97, 82, and 41) and length (bottom, n = 30, 26, 27, and 25) were measured in cells whose shape still covered the entire micropattern after the treatment. Bar, 5 µm. (B) Micropatterned cells were treated with either Y27632 or blebbistatin during starvation. Cells were then fixed and immunolabeled for phosphoezrin (top) and phosphomyosin II (bottom). Z acquisitions were performed and projected on a single image containing the maximal intensity of each pixel. Several images on each micropattern were averaged and color coded with the fire look-up table to highlight intensity variations. Bar, 5 µm. Primary cilium occurrence was measured in each condition (n > 400 for each). (C) RPE1 cells in early G1 were plated at low density on hard substrate (polystyrene-coated glass coverslips) or soft substrate (polyacrylamide gel grafted on glass coverslips). Cells were then serum starved for 48 h, fixed, and stained for F-actin (red), DNA (blue), and acetylated tubulin (green). Most cells on hard substrates had no primary cilium (left). A majority of cells on soft substrates were ciliated (right). Primary cilium occurrence was measured at a cell density of 200 cells/mm2 on hard substrates (n = 300) and on soft substrates (n = 1,140). Primary cilium length was shorter on hard substrates than on soft ones. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Plotted bars represent standard deviations. Horizontal bars represent mean values. Bar, 20 µm.
