Primary ciliogenesis in MDCK cells is governed by constraints in cell area at the single-cell level. (A and B) Simulations (A) and superimposition of the experimental data (dots) shown in Fig. 1 F and the simulations (B). (C–E) A single cell per disk was seeded on disk micropatterns of 700, 1,100, and 1,600 µm2 (C) and incubated for 2 or 4 d to allow the colony to reach a size of four or eight to 12 cells, respectively (D). (D) Cells were then processed for immunofluorescence analysis with antibodies to acetylated tubulin (Ac-tub), MKLP1, and γ-tubulin (γ-tub). Disk micropatterns with 8–12 cells are shown. The green and white arrowheads indicate the midbody remnant and PC, respectively. Nuclei were stained with DAPI. Bars: (C) 40 µm; (D) 6 µm. (E) The percentage of cells with either a midbody remnant or a PC relative to the total number of cells was determined after 2 or 4 d. Data represent the mean + SEM from three independent experiments (n = 180 cells grown for 2 d and n = 524 cells grown for 4 d were analyzed from 45 disk micropatterns each; Student’s t test). (F) The percentage of cells with a peripheral remnant, a central remnant, or a PC relative to the total number of cells was determined after 4 d. (G) The cells at the edge or in internal positions on 700-µm2 micropatterns were analyzed for the presence of peripheral or central midbody remnants or a PC after 4 d. The results are expressed as the percentage of internal or edge cells with peripheral or central remnants or a PC relative to the total number of cells with the corresponding structure. Data in F and G represent the mean + SEM from three independent experiments (n = 176 cells analyzed from 15 disk micropatterns; Student’s t test).
