Inhibition of PP1 targeting to the NE abolishes assembly of lamin B, but not lamins A/C or nuclear membranes. Mitotic HeLa cells were exposed to no peptide, PP1-BD, PP1-BD(V155A), or the transfection reagent DOTAP alone, washed, and released from mitotic arrest for 2 h as in the legend to Fig. 1. (A) Distribution of lamin B, lamins A/C, and LBR was analyzed by immunofluorescence. Arrow points to a PP1-BD–treated cell that probably escaped peptide inhibition and displayed lamin B staining. (B) Dual immunofluorescence labeling of emerin (green) and lamin B (red) in HeLa cells exposed to PP1-BD or PP1-BD(V155A) as in A. Merged images are shown. (C) Proteolysis of lamin B not assembled into the NE. Mitotic HeLa cells were exposed to no peptide, DOTAP alone, PP1-BD, or PP1-BD(V155A) as in A, and extracts were immunoblotted using anti-LBR, anti-AKAP149, anti-lamins A/C, and anti-lamin B antibodies. Δlamin B indicates a 45-kD lamin B fragment. (D) The caspase inhibitor zVAD-FMK prevents proteolytic degradation of unassembled lamin B, but does not rescue nuclear reassembly of lamin B. Mitotic HeLa cells were treated with PP1-BD alone or together with 100 μM zVAD-FMK. Excess peptide was removed and cells were cultured for 2 h with zVAD-FMK. Proteolysis of lamin B and PARP was analyzed by immunoblotting. (E) Distribution of lamin B in cells exposed to PP1-BD or PP1-BD plus zVAD-FMK was examined by immunofluorescence (red). DNA was stained with Hoechst 33342 (blue). Bars, 10 μm.

AKAP149 targets PP1 to the nuclear envelope. (A) PP1, lamin B, and lamins A/C coimmunoprecipitate with AKAP149. NEs were purified from interphase HeLa or KE37 cells, solubilized, and AKAP149 was immunoprecipitated (IP). Control immunoprecipitations were done using preimmune mouse IgGs. Precipitates were analyzed by immunoblotting using anti-AKAP149, anti-PP1, anti-lamin B and anti-lamins A/C antibodies. (B) HeLa cells synchronized in mitosis were incubated with PP1-BD(V155A) or PP1-BD. Excess peptide was removed and cells were cultured for 2 h. Nuclei and NEs were purified, and association of AKAP149 and PP1 with the NE was analyzed by immunoblotting. (C) AKAP149 or PP1 were immunoprecipitated from NEs solubilized from HeLa cells exposed to PP1-BD(V155A) or PP1-BD. Immune precipitates were immunoblotted using anti-AKAP149 or anti-PP1 antibodies.

Cells exposed to PP1-BD undergo apoptosis. (A) Mitotic HeLa cells exposed to PP1-BD or PP1-BD(V155A) were washed and cultured for 6 h. Distribution of LBR, lamin B, lamins A/C, and AKAP149 was examined by immunofluorescence (red label). DNA was stained with Hoechst 33342 (blue) to assess nuclear morphology. (B) Cells treated with PP1-BD or PP1-BD(V155A) were analyzed by immunoblotting using anti-LBR, anti-lamin B, anti-lamins A/C, and anti-AKAP149 antibodies. (C) TUNEL analysis of HeLa cells treated with PP1-BD or PP1-BD(V155A). Fluorescein dUTP label was colored in red and was merged with Hoechst DNA staining (blue) in both panels. (D) Proportions of TUNEL-positive cells over time after exposure to either PP1-BD, PP1-BD(V155A), DOTAP alone or PP1-BD plus 100 μM zVAD-FMK (mean ± SD). Bars, 10 μm.

Synthesis of lamins A/C after inhibition of lamin B assembly by PP1-BD in KE37 cells. (A) Mitotic KE37 cells were exposed to either no peptide, PP1-BD, or PP1BD(V155A), washed, and cultured for 2 h. Distribution of lamins B and A/C was analyzed by dual immunofluorescence. (B) Cells were also analyzed by immunoblotting using anti-lamin B, anti-lamins A/C, anti-LBR, and anti-AKAP149 antibodies. (C) TUNEL analysis of KE37 cells exposed to PP1-BD or PP1-BD(V155A) as in A. Insets, DNA staining with Hoechst 33342. Bars, 10 μm.