FIGURE 6.

LINC complex disruption causes impaired cell migration and polarization. A–C, in vitro scratch wound assay. A, phase contrast images of mCherry and DN KASH expressing MEFs taken at 0 or 3 h post-wound. Scale bar, 80 μm. B, open wound area remaining after 3 h; n = 27 wounds. C, percentage of cells at wound edge with centrosome orientation toward the wound (see scheme) at 0 and 3 h post-wound; n = 127 for mCherry and n = 97 for DN KASH. D–F, cell polarization after culture on micropatterned substrates. D, first panel, red signal, revealing fluorescently labeled fibronectin-coated crossbow pattern and mCherry constructs. Second panel, centrosome labeled by γ-tubulin staining. Third panel, DNA stain. Last panel, merged image; arrows indicate centrosome position. Scale bar, 5 μm. E, percentage of cells with correct polarization. Cells were scored as polarized when the centrosome was located in the forward facing sector (inset, green segment); n = 33 for mCherry and n = 32 for DN KASH. F, average distance of nucleus (Nuc) and centrosome (Cen) from the crossbow pattern center. Control cells display rearward nuclear position and central centrosome position (14); n = 33 for mCherry and n = 32 for DN KASH. G–J, single cell migration analysis of MEFs expressing DN KASH or mCherry. G and H, Rose plots, showing the total distance traveled and the directionality of movement for five representative cells for each cell type during a 6-h period. Average cell speed (I) and persistence time (J) were computed from individual cell traces; n = 138 for mCherry and n = 136 for DN KASH. For each experiment, data are represented as mean ± S.E.; *, p < 0.05.