Extended Data Fig. 10: Modelling nuclear transmigration forces.
From: Compression-dependent microtubule reinforcement enables cells to navigate confined environments
(a) Gradual shrinking of the rear microtubule cushion (grey line) and simultaneous increase of the Rho up-regulation factor (rear contractility multiplier (orange line) after initiation of microtubule cushion disassembly. (b) Velocity of cell (grey) and nucleus (blue) for the simulation in Fig. 6c and Supplementary Video 21 depicting a control cell. The initiation of the Rho burst (red line) accelerates both cell and nucleus which finally leads to the sudden passage of the nucleus through the constriction. (c) Velocity of cell (grey) and nucleus (blue) for the simulation without the microtubule cushion in Fig. 6d and Supplementary Video 21. (d) Disrupting the LINC complex via expression of DN-KASH2 (dominant negative) does not inhibit nuclear passage time in 1205Lu cells. Representative images of 1205Lu cells in constriction microchannels expressing either a KASH2-ext or DN-KASH2 construct. (e) Analysis of nuclear passage times in KASH2-ext and DN-KASH2 expressing cells. Data points represent individual nuclear passages. n = 159 (KASH2-ext) and 209 (DN-KASH2) individual cells pooled from two independent experimental replicates. Statistical analysis by two-tailed Mann-Whitney test. (f) Representative images of control (DMSO) and myosin inhibited (Blebbistatin) 1205Lu cells migrating in confinement. (g) Blebbistatin treatment inhibits cell migration, decreasing cell entry to microchannels. n = the mean number of cells per field of view (FOV) entering microchannels. n = 19 and 20 FOV for control (DMSO) and (-) blebbistatin treatments, respectively. (h) Blebbistatin treatment results in delayed nuclear passage time. n = 41 and 8 cells for control (DMSO) and (-) blebbistatin treatments, respectively. Panel g and h data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. Data shown represents one experiment. Experiments were performed two times with similar results. g, h statistical test by two-tailed Mann-Whitney Test. (i) We use a heuristic to identify occlusion of the passage between the two micropillars (grey) and to find the boundary points between front and rear (see below). This allows to split the volume of the cytoplasm V_0^C into rear and front components V^R and V^F. (j) Sensitivity analysis of the cell response to variations in the threshold pressure at which microtubule (MT) disassembly is triggered. A higher threshold requires more time for the rear pressure to build and trigger the Rho burst and therefore delays transmigration. (k) Time until transmigration vs., gap size in simulations. CLASP1 KD cells only transmigrate when the gap size is large. j and k LOESS regression and 95% confidence intervals (by Bootstrap method) indicated by shaded regions, computed using the R-package spatialeco. Source numerical data are available in Source data.
