Figure 4.
Rapid accumulation of nucleoplasmic LC at the rupture sites. (A) A 488-nm laser is used to nucleoplasmic photobleaching prior to 405-nm laser microirradiation. (B) Side views of before (top left) and after photobleaching (bottom left). Bar: 10 μm. Fluorescence intensity on the white dotted-line arrows along with z-axis was measured and plotted as line intensity profiles (right). (C) Dynamics of mEmerald-LC in response to NE rupture with or without photobleaching the nucleoplasmic pool. The right four columns are magnified views of orange boxes. Top row: A nucleoplasmic area in Lmna-KO MEFs expressing mEmerald-LC (red circle) was photobleached using 488-nm laser, and then a 2-μm spot at the NE (yellow arrowhead) was microirradiated using 405-nm laser during time-lapse imaging with 10 s intervals. Bottom row: The control cells without photobleaching. Bars: 5 μm (the left two columns) and 2 μm (the right four columns). (D) Relative fluorescence intensity of mEmerald-LC at the rupture sites. The mEmerald-LC intensities relative to the initial point are plotted (means ± SEM; n = 20 cells from two independent experiments; **, P < 0.001 from without photobleaching by a linear mixed model). (E–G) Requirements of an NLS for LC accumulation at the rupture sites. mEmerald-LC full-length, ∆417-422 (∆NLS) and ∆417-422 + NLSSUN2 (∆NLS + sunNLS) were expressed in Lmna-KO MEFs and the NE rupture assay was performed as in C and D, without pre-photobleaching. (E) Architecture of the mEmerald-LC NLS mutants. The summary of their dynamics is indicated on the right (+, accumulated at the rupture site; -, not accumulated). (F) Dynamics of mEmerald-LC NLS mutants in response to NE rupture. Bars: 5 μm (the first column) and 2 μm (the second to fifth columns). (G) Relative fluorescence intensities of the mEmerald-LC NLS mutants (means ± SEM; n = 10 cells; **, P < 0.001; ns, P > 0.05 from full-length by a linear mixed model). Full-length (gray) is a reproduction of “Without photobleach” in D.