Journal: Life Science Alliance

Article Title: Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

doi: 10.26508/lsa.202101120

Figure Lengend Snippet: (A) Purified His-lamin A proteins were fractionated by SDS–PAGE and visualized with Coomassie Blue stain. (B) Purified His-lamin A proteins (0.5 mg/ml) stored in the storage buffer with 500 mM NaCl were allowed to polymerize by dialysis in the polymerization buffer with 150 mM NaCl for 3 h at room temperature. His-lamin A proteins were separated into the supernatant (S) and pellet (P) fractions by centrifugation at 14,000 g for 30 min. An equal proportion of His-lamin A proteins were fractionated by SDS–PAGE and visualized with Coomassie Blue stain. The ratio of lamin A polymerization was measured by Image J. Values (means ± SD) are from five independent experiments. (C) Lamin A/C-knockout (LMNA −/− ) HeLa cells were generated by the CRISPR/Cas9 system. An equal amount of the whole cell lysates (WCLs) from LMNA +/+ and LMNA −/− HeLa cells were analyzed by immunoblotting (IB) with antibodies as indicated. (D) LMNA +/+ and LMNA −/− HeLa cells were fixed and stained for lamin A, emerin, and DNA. Representative images are shown. Scale bars, 10 μm. (E) FLAG-lamin A and its mutants were transiently expressed in LMNA −/− HeLa cells. The cells were fixed and then stained for FLAG, lamin B1, and DNA. Representative images are shown. Scale bars, 20 μm. (F) The percentage of FLAG-lamin A transfected cells as described in panel E with nuclear diffusion of FLAG-lamin A was measured (n ≥ 500). *** P < 0.001. (E, G) The fluorescence intensities of FLAG-lamin A in the nucleoplasm and nuclear lamina of the cells as described in panel (E) were measured using ZEISS ZEN2 software. The relative ratios of the signals in the nucleoplasm to the signals in the lamina were calculated. Data are expressed as a ratio relative to FLAG-lamin A WT that set as one. Values (means ± SD) are from at least 50 cells. *** P < 0.001. (E, H) The percentage of FLAG-lamin A transfected cells as described in panel (E) with an elongated nucleus was measured (n ≥ 500). The elongated nucleus is defined as the long nuclear axis is threefold longer than short nuclear axis. Values (means ± SD) are from three experiments. * P < 0.05, ** P < 0.01. (I) LMNA −/− HeLa cells stably expressing FLAG-lamin A or its mutants remained asynchronized (Async.) or were synchronized at the mitosis (M) by treating 200 ng/ml nocodazole for 16 h. An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. (J) LMNA −/− HeLa cells transient expressing FLAG-lamin A or the S22A mutant remained asynchronized (Async.) or were synchronized at the mitosis (M) by treating 200 ng/ml nocodazole for 16 h. FLAG-lamin A was immunoprecipitated with anti-FLAG and the immunonocomplexes were analyzed by immunoblotting with anti-lamin A pY45, anti-lamin A pY45, or anti-FLAG. An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. The Y45 phosphorylation of FLAG-lamin A was quantified and expressed as −fold relative to the level of FLAG-lamin A WT asynchronized. Source data are available for this figure.

Article Snippet: The recovery of the relative fluorescence ratios was normalized to the same region before photobleaching and analyzed using ZEISS ZEN2 image software.

Techniques: Purification, SDS Page, Staining, Centrifugation, Knock-Out, Generated, CRISPR, Western Blot, Transfection, Diffusion-based Assay, Fluorescence, Software, Stable Transfection, Expressing, Mutagenesis, Immunoprecipitation, Phospho-proteomics