Figure 3. Nuclear mDia2 is required for... | Rockefeller University Press

Figure 3.

$Figure 3. Nuclear mDia2 is required for CENP-A levels at centromeres. (A) The mDia2 protein structure showing the relative positions of GBD, DID, FH1, FH2, and DAD domains as well as the position of the NLS. (B) Depletion of mDia2 does not affect CENP-A distribution in the cytoplasm and the nucleus. Cellular fractionation and immunoblotting analysis showing the distribution of CENP-A proteins in the cytoplasm, nucleoplasm, and insoluble materials associated with the chromatin marked by tubulin, RNA polymerase II, and H4K20me2, respectively. A higher exposure of CENP-A blot was shown for its cytoplasmic distribution. (C) Cells expressing wild-type mDia2 or the mDia2K35A/R36A mutant (with a defective NLS) fused with mEmerald were treated with or without LMB (20 nM for 60 min) before fixation and imaging mEmerald. Bar, 10 µm. (D) Immunofluorescence detection of CENP-A and CENP-B 48 h after transfection with the indicated siRNAs and the full-length mDia2 (FL mDia2) or mDia2 ΔNLS mutant expression vectors (DNA - DAPI). Bar, 10 µm. (E) Quantification showing the normalized CENP-A integrated intensity per nucleus (mean ± SD overlaid with scatterplot). The p-value was computed using a two-tailed t test. Control: n = 174, mDia2 siRNA: n = 149, mDia2 siRNA + FL-mDia2: n = 136, and mDia2 siRNA + FL-ΔNLS: n = 103.$

Nuclear mDia2 is required for CENP-A levels at centromeres. (A) The mDia2 protein structure showing the relative positions of GBD, DID, FH1, FH2, and DAD domains as well as the position of the NLS. (B) Depletion of mDia2 does not affect CENP-A distribution in the cytoplasm and the nucleus. Cellular fractionation and immunoblotting analysis showing the distribution of CENP-A proteins in the cytoplasm, nucleoplasm, and insoluble materials associated with the chromatin marked by tubulin, RNA polymerase II, and H4K20me2, respectively. A higher exposure of CENP-A blot was shown for its cytoplasmic distribution. (C) Cells expressing wild-type mDia2 or the mDia2^K35A/R36A mutant (with a defective NLS) fused with mEmerald were treated with or without LMB (20 nM for 60 min) before fixation and imaging mEmerald. Bar, 10 µm. (D) Immunofluorescence detection of CENP-A and CENP-B 48 h after transfection with the indicated siRNAs and the full-length mDia2 (FL mDia2) or mDia2 ΔNLS mutant expression vectors (DNA - DAPI). Bar, 10 µm. (E) Quantification showing the normalized CENP-A integrated intensity per nucleus (mean ± SD overlaid with scatterplot). The p-value was computed using a two-tailed t test. Control: n = 174, mDia2 siRNA: n = 149, mDia2 siRNA + FL-mDia2: n = 136, and mDia2 siRNA + FL-ΔNLS: n = 103.