Figure 2.
Active remodeling of cytoskeleton networks and RAC1-mediated dynamic cell-cell connections lead to collective NSF
(A) Representative fluorescent images of ICM during periodic TNF-α stimulation show that shape transition of the nuclear (green) and actin deformation (red) are more dramatic at the marginal region. Scale bar represents 20 μm.
(B) Averaged extension of actin filaments at different time points reveals that actin deformation differs at the marginal and interior area. The variations in the average actin extension were normalized by the initial value (i.e., at 0 min).
(C) Cross-correlation analysis between variations in the volume of microtubule networks and NSF illustrates that remodeling of microtubule networks has trivial effects on nuclear shape. The error bars represent standard deviation of the correlation coefficients between individual cell's microtubule remodeling and NSF.
(D) Cross-correlation analysis between variations in actin extension and NSF reveals that actin deformation regulates nuclear shape. The error bars represent standard deviation of the correlation coefficients between changes in individual cell's actin extension remodeling and NSF.
(E) Representative fluorescent images of the actin filaments at cell-cell connections during periodic TNF-α stimulation show variations in the fluorescence intensity. Scale bar represents 20 μm.
(F) Traces of the fluorescence intensity at the cell-cell connections demonstrate that the variations are more obvious with TNF-α stimulation. The variations in the average actin extension were normalized by the initial value (i.e., at 0 min).
(G) Counts of the events with fluctuation amplitude of fluorescence intensity at cell-cell connections more than 10% show more frequent loss of cell-cell contacts with TNF-α stimulation. The error bars represent standard deviation of cell-cell connection loss counts among low density cells and in ICM.
(H and I) Expression level of RhoA (H) and RAC1 (I) mRNA detected by RT-PCR and expressed as fold-change. For the data presented in (H) and (I), minimum five independent experiments were performed for each data point. The expression level of both proteins was normalized by the value of control samples, i.e., the untreated ICM. The error bars represent standard deviation of protein expression level in five independent experiments at each condition.
(J) Cross-correlation analysis of the NSF of neighboring cells reveals that the collective cellular responses are disrupted by drugs regulating cell-cell connections. Correlation coefficients obtained under different conditions were normalized to the control sample, i.e., ICM treated by 1/20 min−1 TNF-α. The error bars represent standard deviation of the NSF correlation coefficients among population cells.
(K) Schematic shows that entrainment in the Rho-associated signaling pathways leads to an elevated RAC1 expression level, which facilitates transition to more dynamic cell-cell connections.
(L) Schematic shows that deformation of actin filaments leads to changing mechanical loads on the nucleus.