Figure 7.
Dynamics of LC, BAF, and cGAS in response to laser microirradiation-induced NE rupture. (A and B) Dynamics of sfGFP-DARPin-LA6, Halo-BAF, and cGAS-sfCherry in response to NE rupture. cGAS-sfCherry was localized to the cytoplasm in some cells (A) or to the nucleus in the others (B). The right four columns are magnified views of orange boxes, and the rupture sites are indicated with yellow arrowheads. (C) Normalized fluorescence intensities of sfGFP-DARPin-LA6, cGAS-sfCherry and Halo-BAF in MEFs are shown up to the signal peaks, and cGAS-sfCherry is localized to the cytoplasm (top) or the nucleus (bottom; means ± SEM; n = 10 cells; **, P < 0.001 from others by a linear mixed model). (D) Representative images of single confocal sections of MEFs fixed within 10 min after laser microirradiation and stained with anti-cGAS, Alexa Fluor 488-labeled anti-rabbit IgG, and Hoechst 33342 for DNA. Colored arrowheads indicate sites of NE rupture induced by laser microirradiation in cells with cytoplasmic cGAS (orange) or nuclear cGAS (blue). Bar: 10 μm. (E) Fluorescence intensities of the cGAS accumulated at the rupture sites was measured. (n = 15 and 8 for cytoplasmic and nuclear cGAS, respectively from two independent experiments; horizontal dotted lines show the mean values, *, P < 0.05 from cells with the accumulation of cytoplasmic cGAS by a Mann-Whitney U test). (F) Maximum intensity Z-projection of high-resolution confocal images of LC, BAF, and cGAS in a NE protrusion from the nuclear main body. The sfGFP-DARPin-LA6 expressing cells are fixed within 10 min after laser microirradiation, stained with anti-BANF1 (3F10-4G12) and anti-cGAS, followed with Cy5-labeled anti-mouse IgG and Alexa Fluor 568-labeled anti-rabbit IgG, respectively, and Hoechst 33342 for DNA. Magnified views of an orange boxed area (top left) are shown as merged and individual images. Merged images (top left and top middle) show LC (blue), BAF (red), and cGAS (green). (G) Line intensity profiles over the NE protrusion. Fluorescence intensity on the white dotted-line arrow was measured and plotted. (A, B, and F) Bars: 5 μm (A and B, the first column; and F, the top left); and 2 μm for the magnified views.