A Par3-regulated retrograde movement of actin structures toward the nucleus correlates with NE rupture. (A) Video stills taken from 3D-cell imaging of representative U2OS cells treated with siCTL or siPar3, expressing E4orf4-mCherry and the actin marker LifeAct-GFP, showing the movement of actin structures over time relative to E4orf4 nuclear signal, as pointed by colored arrowheads; actin filaments move directionally toward the nucleus in siCTL-treated cells, whereas they move randomly in siPar3-treated cells. Bar: 20 µm. See also Videos 7, 8, and 9. The dashed lines identify the cross sections perpendicular to actin network at t = 0 min. (B) Corresponding kymographs of actin structure dynamics (in pseudo-color); E4orf4 signal along the cross sections is shown as a reference (in gray), and the arrow designates the time of NE rupture in a siCTL-treated cell. (C) Quantification of actin structure velocity toward the nucleus in E4orf4-expressing cells, as measured from the probability distribution from all the visible actin structure velocity distributions; siCTL, ●, n = 9 cells; siPar3, ○, n = 8 cells. The plain lines provide the Gaussian fit of the normal distribution. Actin structures moving toward the nucleus were defined as having a positive speed, whereas those moving away as having a negative speed. (D) Linear plot of the time of nuclear rupture as a function of the speed of actin structures toward the nucleus, showing a linear relationship (R2 = 0.8945); each data point corresponds to the average actin speed ± SD and rupture time ± 2.5 min (e.g., half the acquisition rate) for a single cell, and the shaded area provides the 95% CI of the linear regression. *, P < 0.05.
