Figure 3.

Nuclear flattening does not require actomyosin contraction. (A) Images show cells pretreated with drug for 1 h, trypsinized, and then seeded onto substrates for 1 h in the presence of the drug. Cells in the presence of Y-27632 and blebbistatin showed clear effects on the cell morphology compared with the control, but the nucleus was still flattened as evident from the x-z cross section. ML-7 treatment on the other hand prevented the spreading of the cell as well as the flattening of the nucleus. Scale bar for x-y views is 20 μm and for x-z views is 5 μm. (B) Nuclear aspect ratio was larger in ML-7 treated cells reflecting unflattened nuclei, consistent with the fact that the cells were unable to spread in presence of ML-7 (C) Minor differences in aspect ratio on Y-27632 or blebbistatin treatment reflect minor effects on degree of cell spreading. (D) Aspect ratio correlates with the degree of cell spreading. In ML-7 treated cells, the cells were unable to spread (blue diamonds) corresponding to the large aspect ratio; none of the other treatments prevented nuclear flattening but concomitantly, they did not prevent cell spreading (n$\ge$ 31 for all conditions, ^∗p < 0.05; all comparisons are with untreated controls). The experiments in (E)–(H) show the corresponding results on treating well-spread cells (cultured overnight on fibronectin-coated glass-bottom dishes) with myosin inhibitors. Blebbistatin treatment rounded up spread cells, and caused rounded nuclear shapes. Images are shown in (E), whereas the scatter plots of aspect ratio versus cell spreading area are shown in (H). (F) and (G) show the average aspect ratio and areas for the different myosin inhibitors (n$\ge$ 24, ^∗p < 0.05; all comparisons are with untreated control). Scale bar in (H) is 20 μm for the x-y view and 5 μm for the x-z view. All data are shown as mean $\pm$ SEM. To see this figure in color, go online.