Figure 5.
Effect of LC Ig-fold laminopathy mutations and BAF-KD on accumulation kinetics of LC at the rupture sites. (A–G) The NE rupture assay was performed with mEmerald-LC mutants in Lmna-KO MEFs (A–E) and sfGFP-DARPin-LA6 in BAF-KD MEFs (F and G). (A) Architecture of mEmerald-LC full-length, ∆432-572 (∆Tail) and ∆433-548 + Ig-foldLB1 (∆IgF + b1IgF). The summary of their dynamics is indicated on the right (+, accumulated at the rupture site; -, not accumulated). (B) Dynamics of mEmerald-LC Ig-fold mutants in response to NE rupture in Lmna-KO MEFs. (C) Relative fluorescence intensities of the mEmerald-LC Ig-fold mutants (means ± SEM; n = 10 cells; **, P < 0.001 from full-length by a linear mixed model). (D) Positions of laminopathy mutations in the LA/C Ig-fold structure (PDB accession no. 1IFR). The amino acid residues whose mutations affect BAF binding affinity in vitro (Samson et al., 2018) are colored (red, no detectable binding; orange and magenta, very weak binding; and purple and blue, ∼fivefold weaker binding to the WT). The two residues whose mutations have no effect on BAF binding affinity in vitro are shown in dark green and light green. (E) Relative fluorescence intensities of the mEmerald-LC Ig-fold laminopathy mutants in Lmna-KO MEFs (means ± SEM; n = 10 cells; **, P < 0.001 from full-length by a linear mixed model). See Fig. S4 A for microscopic images. (F) Dynamics of sfGFP-DARPin-LA6 in response to NE rupture in WT MEFs expressing shRNAs, scrambled control (shScr), shBAF#1 or shBAF#2 (see Fig. S4, B and C for the validation of KD by immunofluorescence and immunoblotting). (G) Relative fluorescence intensities of sfGFP-DARPin-LA6 in the indicated cells (means ± SEM; n = 10 cells; **, P < 0.001 from the shScramble by a linear mixed model). (C and E) Full-length (gray) is a reproduction of “Without photobleach” in Fig. 4 D. (G) shScr (gray) is a reproduction of “shScramble” in Fig. 2 F. (B and F) The right four columns are magnified views of orange boxes. Bars: 5 μm (the first column) and 2 μm (the second column to others).

Effect of LC Ig-fold laminopathy mutations and BAF-KD on accumulation kinetics of LC at the rupture sites. (A–G) The NE rupture assay was performed with mEmerald-LC mutants in Lmna-KO MEFs (A–E) and sfGFP-DARPin-LA6 in BAF-KD MEFs (F and G). (A) Architecture of mEmerald-LC full-length, ∆432-572 (∆Tail) and ∆433-548 + Ig-foldLB1 (∆IgF + b1IgF). The summary of their dynamics is indicated on the right (+, accumulated at the rupture site; -, not accumulated). (B) Dynamics of mEmerald-LC Ig-fold mutants in response to NE rupture in Lmna-KO MEFs. (C) Relative fluorescence intensities of the mEmerald-LC Ig-fold mutants (means ± SEM; n = 10 cells; **, P < 0.001 from full-length by a linear mixed model). (D) Positions of laminopathy mutations in the LA/C Ig-fold structure (PDB accession no. 1IFR). The amino acid residues whose mutations affect BAF binding affinity in vitro (Samson et al., 2018) are colored (red, no detectable binding; orange and magenta, very weak binding; and purple and blue, ∼fivefold weaker binding to the WT). The two residues whose mutations have no effect on BAF binding affinity in vitro are shown in dark green and light green. (E) Relative fluorescence intensities of the mEmerald-LC Ig-fold laminopathy mutants in Lmna-KO MEFs (means ± SEM; n = 10 cells; **, P < 0.001 from full-length by a linear mixed model). See Fig. S4 A for microscopic images. (F) Dynamics of sfGFP-DARPin-LA6 in response to NE rupture in WT MEFs expressing shRNAs, scrambled control (shScr), shBAF#1 or shBAF#2 (see Fig. S4, B and C for the validation of KD by immunofluorescence and immunoblotting). (G) Relative fluorescence intensities of sfGFP-DARPin-LA6 in the indicated cells (means ± SEM; n = 10 cells; **, P < 0.001 from the shScramble by a linear mixed model). (C and E) Full-length (gray) is a reproduction of “Without photobleach” in Fig. 4 D. (G) shScr (gray) is a reproduction of “shScramble” in Fig. 2 F. (B and F) The right four columns are magnified views of orange boxes. Bars: 5 μm (the first column) and 2 μm (the second column to others).

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