Nucleo-cytoplasmic shuttling of NPM1wt and NPM1 mutants. (A) Physiological NPM1wt shuttling. The bipartite NLS drives the nuclear import of NPM1wt. The C-terminal 3-helix NoLS dictates NPM1wt positioning within the nucleolus, whereas the 2 weak nuclear export signals (NESs) at the N terminus are responsible for its export from the nucleus to the cytoplasm (dashed arrow). Eventually, the nuclear import predominates over export so that almost all NPM1wt resides in the nucleolus. (B) Abnormal traffic of NPM1 mutants. Changes in tryptophans (W) 288 and 290 (or 290 alone), with consequent unfolding of the C-terminal triple-helix, abrogate the capability of the mutant to reside within the nucleolus. Furthermore, the insertion of a third NES motif at the C terminus markedly increases the export of the protein (thick arrow). Thus, the nuclear export predominates over the import, and the NPM1 mutant is delocalized in the cytoplasm. The 3-dimensional reconstruction of confocal images shown in the circles has been adapted from Falini et al.⁷  Nuclei are stained with 4′,6-diamidino-2-phenylindole, while the nucleoli are double-stained for NPM1 and nucleolin. Boxes show OCI-AML3 cells engineered to express either endogenous NPM1wt or NPM1 mutant fused to GFP. Nuclei are stained with Hoechst 33342.