Figure S2.
Cytokinesis Cells Exhibit Higher Mechanical Resistance Than Interphase Cells in Distinct Cellular Regions; High-Resolution AFM Images on Supported Lipid Bilayers Reveal a Complex Distribution of Phases, Related to Figure 2
(A) Typical force-distance curve conducted on a live dividing HeLa cell in the nuclear area. This event is distinguishable by six consecutive discontinuities corresponding to the breakthrough of the plasma membrane (two events) in addition to those of the nuclear envelope (composed by two lipid bilayers and therefore showing four breakthrough events).
(B) Scatterplot of the upper membrane breakthrough force (first event) versus the cell height for interphase (blue) and cytokinesis cells (yellow) in the nuclear area (19 cells per case). As in the case of the cytoplasmic region, the breakthrough force steadily increases with the cell height. When selecting a comparable range of cell heights (4.1-8.3 μm), the mechanical resistance of the nuclear region in cytokinesis membranes is clearly higher (174 ± 42 nN, n = 34) than that of nondividing cells (76 ± 19 nN, n = 29) (inset in B) with a > 99.99% confidence (Student’s t test).
(C–I) Supported lipid bilayers isolated from live cells and analyzed at high-spatial resolution by AFM imaging exhibit a complex distribution of phases and mechanical stabilities. While Figure 2 D–E reflects the most distinctive patterns of phase distribution in our supported lipid bilayer samples, in some of the preparations additional phase distributions were observed. (C) Pie chart corresponding to S phase preparations shows that, while samples showing only a P0 phase are predominant (Figure 2D), in some cases P0 is accompanied by P1 or P2 phases. Note that the nomenclature defining each of the phases is arbitrary, yet self-consistent throughout the paper. (D) In the case of lipid bilayers formed from cytokinesis cells, a wider combination of the four distinct observed phases is encountered, as shown in the pie chart. The very stiff P3 phase was never observed in lipid bilayers isolated from S phase cells. Each pie chart contains 10 and 14 sets of experiments for S phase and cytokinesis cells, respectively. Particularly interesting phase combinations that are not discussed in the main text for cytokinesis preparations are shown in E and F. (E) A P1 phase that is only slightly higher than the matrix P0 phase (<0.2 nm) and readily apparent in the phase channel (not shown). (H) Notably, there is no measurable difference in the mechanical stability of the P0 and P1 phases (3.7 ± 1.7 nN). (F) Another common combination of phases is observed in F, whereby only P0 and P2 phase coexist. In this case, the height of the P2 phase is significantly higher (>1.27 nm) than the matrix P0 phase. (I) The mechanical stability of the P2 phase is higher (8.0 ± 1.3 nN) than the P0 phase (3.7 ± 0.6 nN).
A larger version of Figure 2E with the associated height profile is shown in G. In this case three main phases are observed: a first P0 phase exhibiting 2.86 ± 0.52 nm, a second phase (P2) showing a height of 4.13 ± 0.54 nm relative to the mica substrate, and a higher P3 phase, featuring 4.96 ± 0.16 nm. For S phase cells, an average height of 2.63 ± 0.72 nm is observed for the matrix, continuous P0 phase (ref. to Figure 2D, height profile not shown).