Figure 1.
Differential nuclear dynamics of enhanced green fluorescence (EGFP)–Top1 variants in live cell. (A) Schematic representation of human Top1 organized into four domains: N-terminal domain (1–214 aa), core (215–635 aa), linker (636–712 aa) and C-terminal domain (713–765 aa) based on the crystal structure(2,13). N-terminal domain harbours tryptophan anchor (W203, 205, 206), C-terminal domain harbours catalytic active site (Y723) and CPT interacting site (N722) are also shown. (B) Expression of EGFP–Top1 was unaffected with indicated mutations. Immunoblotting of HCT116 cells expressing ectopic EGFP–Top1WT (lane 1), EGFP–Top1Y723F (lane 2), EGFP–Top1N722S (lane 3), EGFP–Top1W205G (lane 4) and EGFP empty vector [VC] (lane 5). Blots were probed with antibodies against GFP (top) or Actin as loading control (below) (C) EGFP-linked and endogenous Top1 are indicated on the extreme right probed with human Top1 antibody. (D) Representative images showing colocalization of the ectopic EGFP–Top1WT (green) with the nucleoli (red). EGFP–Top1 WT construct was expressed in HCT116 cells in the absence (Ctr) or presence of CPT (10 μM for 30 min). Immunofluorescence staining of EGFP–Top1 with anti-GFP antibody (green) and anti-nucleolin antibody (red) was done. Cells were counterstained with DAPI to visualize nuclei. (E) Representative images showing differential nucleolar localization of EGFP–Top1 variants in the presence of camptothecin (CPT). All the EGFP–Top1 constructs were separately expressed in HCT116 cells and were imaged 24 h post-transfection under live cell confocal microscopy. Cells were treated with CPT (10 μM for 30 min) as indicated. Nuclei were stained with Hoechst 33342 (blue). (F) Densitometry analysis of CPT-induced delocalization of EGFP–Top1 variants (shown in panel E) from the nucleolus to the nucleoplasm. The percentage of cells displaying nucleolar fluorescence was determined from at least 60–70 cells expressing individual EGFP–Top1 constructs (WT, Y723F, N722S, W205G) in the presence and absence of indicated drugs. Error bars represent mean ± S.E. (n = 70).