Fig. 4 | Nature Communications

Fig. 4

From: LINC complex-Lis1 interplay controls MT1-MMP matrix digest-on-demand response for confined tumor cell migration

Fig. 4

Lis1 contributes to nucleus-centrosome linkage in MDA-MB-231 cells. a Schematic representation of MDA-MB-231 cell expressing H2BmCherry and with SiR-tubulin-labeled microtubules migrating through the microfabricated channel. Centrosome displacement was measured before (pre) and after (post) irradiation with the laser beam (blue dots) focused between the nucleus and the centrosome. Dashed green circles, positions of the centrosome before laser irradiation; green dot, centrosome position after irradiation. For each cell analyzed (n = 18), the graph shows paired mean centrosome displacements (in μm) before (pre) and after (post) irradiation; Wilcoxon signed rank test. b Kymographs showing elastic recoil of the centrosome after laser irradiation (time 0). H2BmCherry-labeled nucleus is shown in red, SiR-tubulin-labeled microtubules and the centrosome are in green. c Lis1 and Nesprin-2 immunostaining in MDA-MB-231 cells treated with nocodazole. d MDA-MB-231 cells treated or not with nesprin-2 siRNA were treated with nocodazole and immunostained for Lis1 and LMNA. Insets in c and d represent Lis1 signal of the boxed regions. Arrowheads point at regions of Lis1 association with the NE. e Percentage of cells with Lis1 association with the NE was scored in cells treated with indicated siRNA. n, number of cells analyzed from three independent experiments; Fisher exact test. f Pericentrin and DAPI immunostaining. Scale bars=5 μm. Mean centrosome-nucleus distance (μm) in MDA-MB-231 cells in 3D collagen under indicated conditions ± SEM; n, number of cells analyzed from three (siLis1 and siNesprin-2) or two (siLMNA) independent experiments; Kruskal–Wallis test. g Centrosome position related to the nucleus was scored as schematized from time-lapse sequences of MDA-MB-231 cells expressing GFPcentrin-1 and H2BmCherry and treated with Lis1 siRNA or with control siNT during invasion through the small pore size collagen gel. The graph represents the percentage of frames with the centrosome ahead of the front edge of the nucleus for each cell analyzed and the corresponding Box-and-whisker plot. n, number of cells analyzed from three independent experiments; Mann-–Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars=10 μm

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