Figure 2. Changes in nuclear import and export are specific to MKL1 and are caused by altered actin dynamics in Lmna^−/− and Lmna N195K cells.

(a, b) Change in nuclear fluorescence intensity over time upon serum stimulation in Lmna^+/+, Lmna^−/− and Lmna N195K MEFs expressing MKL1-GFP in the absence (a) or presence of leptomycin B (b). Values were normalized to the initial nuclear fluorescence intensity before serum addition. N = 20 for each cell line. (c) Fluorescence loss in photobleaching (FLIP) experiments of MKL1-GFP to measure nuclear export. Increased loss of nuclear fluorescence indicates a higher rate of nuclear export of MKL1-GFP in lamin mutant cells. N = 10 for each cell line. (d) Schematic representation (not drawn to scale) of full length MKL1-GFP (top) and MKL1(1–204)–2×GFP (bottom), consisting of the N-terminal actin binding domain of MKL1 fused to two GFP moieties. RPEL motifs depicted in blue and red, NLS in yellow, DNA binding domain (SAP) and transcriptional activation domain (TAD) in light blue, coiled-coil domain in purple, other parts of the C-terminus in dark blue. (e) Representative frames from time-lapse series of Lmna^+/+, Lmna^−/− and Lmna N195K MEFs expressing MKL1(1–204)–2×GFP following serum stimulation. Scale bar, 10 μm. (f) Lmna^+/+ MEFs showed rapid accumulation of MKL1(1–204)–2×GFP in the nucleus upon serum stimulation, whereas nuclear accumulation was slower in Lmna^−/− and Lmna N195K cells. Nuclear fluorescence intensity was normalized to the initial nuclear fluorescence before serum stimulation. N = 60 for each cell line. (g) FLIP experiments in cell expressing MKL1(1–204)–2×GFP. Fluorescence intensity values were normalized to the initial nuclear fluorescence intensity before bleaching of a cytoplasmic region. N = 10 for each cell line. Error bars, s.e.m.