Mechanism of perturbed NPM nucleocytoplasmic traffic in NPMc⁺ AML. (A) Cytoplasmic accumulation of NPM is NES-dependent. OCI-AML3 cell line carrying the NPM1 mutation A shows cytoplasmic (and nucleolar) positivity for NPM (green) (top, left). NPM is relocated into the nucleus after exposure to the Crm1 inhibitor leptomycin B (top, right). Cytoplasmic accumulation of NPM is tryptophan dependent. In NIH-3T3 cells, NPM mutant A is relocated from cytoplasm (green, bottom, left) into nucleoli (green, bottom right), after the 2 tryptophans (W) are reinserted at positions 288 and 299. Images were collected with a Zeiss LSM 510 confocal microscope using a Plan Apochromat 100×/1.4 NA oil objective and LSM 5 software for image acquisition. Nuclei were stained with propidium iodide (Molecular Probes). Three-dimensional reconstruction of the confocal images and electronic cuts of cells were performed with Imaris software. (B) Hypothetical mechanism of altered nucleocytoplasmic traffic of NPM mutant A and NPMwt. Green boxes indicate NPMwt protein; blue boxes, mutated NPM protein; red circles, tryptophan residues; black circles, mutated tryptophans; magenta boxes, NES motif; and NLS, nuclear localization signal.