Figure 1

Substrate adhesion controls the symmetry of the cleavage furrow.

(A) XY and XZ views of the cleavage furrow of a HeLa cell cultured on 10 μg/mL FN and stained for endogenous NMIIA and DNA. (B) XZ views of the cleavage furrow of cells cultured on low (1 μg/mL) and high (50 μg/mL) FN substrates. XZ projections were made from a similar sized ROI as in (A). Ingression from the bottom (double headed green arrow) was measured as the distance between the substrate (dotted yellow line) and the bottom of the cleavage furrow. Cells were grouped based on the height of the cleavage furrow into early (>15 μm), mid (9–15 μm) and late (3–9 μm) stages of ingression. Measurements were made on 34 cells and 42 cells for 1 μg/mL and 50 μg/mL FN, respectively, across 6 independent experiments for each condition (see Methods). (C) XY views of HeLa cells at anaphase stained for paxillin, cultured on low and high adhesive substrates. Look up table is fire and color bars show the gray scale values of the images. White arrows show retraction fiber adhesions and green arrows show mitotic FAs. (D) Merged XZ views of HeLa cells at anaphase stained for paxillin (green) and NMIIA (gray) cultured on low and high adhesive substrates. XY views are shown in Figure S1C. (E) TIRF time montage of a HeLa cell expressing Paxillin-mEGFP and H2B-mCherry cultured on high adhesive substrate undergoing anaphase imaged using TIRF microscopy. Ingression starts at 0 min and the arrowheads indicate the position of the cleavage furrow. Arrows denote the side with larger adhesions maintained until the daughter cells start spreading at 10 min. (F) Quantification of relative paxillin intensity comparing adhesions underneath the cleavage furrow (red ROI in inset) and immediately adjacent to the cleavage furrow (blue ROI in inset). Measurements were made from 7 cells across 5 independent experiments. (G) Kymograph created from blue line in (C). Dotted line denotes the onset of ingression. * denotes p < 0.05 and ** denotes p < 0.01; Scale bars, 5 μm. Error bars show standard error of the mean (SEM).