Supplemental material JCB. Pitaval et al., Cell shape and ciliogenesis Pitaval et al.

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1 Supplemental material THE JOURNAL OF CELL BIOLOGY Pitaval et al., S1

2 Figure S1. Cell cycle exit analysis. (A) Experimental procedure. RPE1 cells were synchronized at the G1/S transition and released with complete medium before being starved at various time points along the cell cycle in separated distinct experiments in a micropatterned 12-well plate. Cell behaviors were followed in time-lapse video microscopy. (B) Cells were plated on large, 500-µm-wide, fibronectin-coated discoidal micropatterns and monitored in time-lapse phase-contrast microscopy over 54 h to measure cell division time with respect to the time of starvation and to observe daughter cell fates. A few examples are highlighted. The blue cell divided once in the presence of serum. The daughters were starved in late G1 and gave rise to four daughters over the entire experiment. The red cell divided once in the presence of serum. The daughters were starved in early G1 and didn t divide any longer (Video 1). Bar, 100 µm. (C) Quantification of cell behavior upon serum starvation. The respective proportions of nondividing cells (red), cells dividing once (light blue), and cells dividing more than twice (dark blue) are shown on the histogram. The left bar shows cells that were not starved, and most cells divide several times (n = 16). In the middle bar, cells were starved in S phase, and in the right bar, cells were starved in G2 phase (n = 31 in both conditions). In these two cases, most cells divided once and then left the cell cycle. (D) Quantification of cell behavior upon serum starvation at various time points in G1. All cells divided once in the presence of serum and were starved in G1. The histogram shows the proportion of dividing cells (blue) and nondividing cells (red) depending on the duration between cell entry into G1 phase and the serum starvation time (n = 44, 46, 26, 46, 20, 36, and 28, respectively). We observed a progressive reduction of cells exiting the cell cycle all along G1. Plotted bars represent standard deviations. S2

3 Figure S2. Ezrin and myosin II localization and density. (A) RPE1 cells on small (750 µm2) and large (3,000 µm2) micropatterns were 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. Grayscales are identical for cells on small and large micropatterns. Ezrin staining reveals the presence of small microvilli only in confined cells. Myosin II staining decorates actin bundles in confined and spread cells, but these bundles are more conspicuous and numerous in spread cells. Bar, 5 µm. (B) RPE1 cells were plated on small or large micropatterns and treated or not treated with 10 µm Y27632 in the absence of serum for 24 h. Cells were fixed and stained for phosphoezrin and phosphomyosin II. The mean intensity per pixel (after background subtraction) was measured on individual images and compared between the two micropattern sizes. Ezrin staining was more intense in confined than in spread cells (top). Y27632 treatment slightly reduced phosphoezrin densities in confined but not in spread cells (middle). Phosphomyosin II staining appeared more dense in spread than in confined cells. Y27632 treatment strongly reduced phosphomyosin II densities in both confined and spread cells (bottom). a.u., arbitrary units. (C) Confocal Z stack optical sections showing dense phosphoezrin staining on top of confined cells (left) and dispersed staining on top of extended cells (right), in which the basal concentration of actin fibers is higher than on small cells. Bar, 5 µm. (D) RPE1 cells were plated on hard (polystyrene) and soft (polyacrylamide) substrates. They were starved for 24 h and stained for F-actin (left), phosphomyosin (middle), and acetylated tubulin (in white in the merge picture on the right). DNA is in blue. Actin bundles appeared thicker and straighter, and therefore probably more contracted, on hard than on soft substrate. *, P < 0.05 and ***, P < Horizontal bars represent mean values. Bars, 20 µm. S3

Figure S3. Labeling and quantification of centrosome position. RPE1 cells were plated on small (750 µm2) and large (3,000 µm2) micropatterns in the absence of serum for 24 h.

4 Figure S3. Labeling and quantification of centrosome position. RPE1 cells were plated on small (750 µm2) and large (3,000 µm2) micropatterns in the absence of serum for 24 h. (A) Micropatterned cells were fixed with paraformaldehyde. They were then immunolabeled for acetylated tubulin to reveal the primary cilium and/or cytoplasmic acetylated microtubules (green), immunolabeled for phosphomyosin II to detect the centrosome/basal body (red), and then stained with phalloidin to reveal actin filaments (blue) and Hoechst to reveal the nucleus (not depicted). The top row shows a primary cilium and the basal body on the dorsal surface of a cell confined on a small micropattern. The bottom row shows the acetylated microtubule network and the centrosome on the ventral surface of a cell spread on a large micropattern. Bars, 15 µm. Inset bars, 3 µm. (B) Micropatterned cells were fixed with cold methanol. They were then immunolabeled for -tubulin to reveal microtubules (red), immunolabeled for -tubulin to reveal centrosomes (green), and stained with Hoechst to reveal nucleus (blue). Z acquisitions were performed to detect the position of the centrosome (green arrows). It was either found above the nucleus, close to the dorsal cell surface (top), or below the nucleus, close to the ventral cell surface (bottom). Bars, 15 µm. The proportion of centrosome/basal body in the apical position close to the dorsal cell surface was quantified in cells plated on small (top, 750 µm2) and large (bottom, 3,000 µm2) micropatterns in the presence of culture medium without serum (control) and various cytoskeletal inhibitors (750 µm2: control, n = 84; cytochalasin D, n = 63; Y27632, n S4

5 Video 1. Monitoring of cell fate upon serum starvation. RPE1 cells were plated on large, 500-µm-wide, fibronectin-coated discoidal micropatterns and monitored in 10 time-lapse phase-contrast microscopy to measure cell division time with respect to the time of starvation and observe daughter cell fates. Time interval between two frames is 10 min. Colored dots are used as visual marks to follow individual cell fates. The blue cell s daughters were starved in late G1 and divided again. The red cell s daughters were starved in early G1 and didn t divide again. The green cell s daughters adopted asymmetric behaviors: one divided again and not the other. = 101; and 3,000 µm 2 : control, n = 49; cytochalasin D, n = 36; Y27632, n = 98). (C) Micropatterned cells were fixed with paraformaldehyde. They were then immunolabeled for acetylated tubulin (green), stained with phalloidin to reveal actin filaments (red), and stained with Hoechst to reveal nucleus (blue). Confocal Z stacks were performed and were either projected on the XY plane or sectioned along the XZ plane. Side views show the location of primary cilium on top of confined cells (left) and the presence of acetylated microtubules instead of a primary cilium in the cytoplasm of extended cells (right). X bars, 5 µm. Z bar, 2 µm. (D) The proportion of ciliated cells (Fig. 4) after different treatments is plotted against the proportion of cells having their centrosome in an apical position, close to the dorsal cell surface (see B). The two parameters could be linearly correlated with a correlation factor r 2 = Plotted bars represent standard deviations. S5

T H E J O U R N A L O F C E L L B I O L O G Y

T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Rodríguez-Fraticelli et al., http://www.jcb.org/cgi/content/full/jcb.201203075/dc1 Figure S1. Cell spreading and lumen formation in confined