Fig. 2
Nuclear accumulation of Polo depends on an NLS that is masked by the PBD. a Alignment of Polo sequences from different species reveals a conserved bipartite NLS in the KD comprising the NLS1 and NLS2 motifs. b, c Time-lapse imaging of stable cell lines expressing PoloNLS7A-GFP and PoloT182D, NLS7A-GFP with RFP-Lamin. Bar: 5 μm. d The GFP fluorescence ratio (nucleus/total cell) was measured in the period preceding NEB (n = 10, error bars: SD). e A syncytial embryo expressing PoloNLS7A-GFP was observed by time-lapse microscopy. N: a nucleus; arrow: a centrosome; arrowhead: midbody ring. Bar: 10 μm. f The NLS in the crystal structure of a complex between the KD (green) and the PBD (cyan) of zebrafish Plk1 and the inhibitory peptide from Drosophila Map205 (red) (Protein Data Bank accession no. 4J7B17). The surface area surrounding NLS1 and NLS2 residues is shown (dark green) to highlight their interaction with the PBD. Close-ups show the interface between NLS residues and the PBD (top) and the same surface with a clockwise 90° rotation (bottom). g Dashed boxes emphasize details of the interaction between NLS1 and a PBD peptide in zebrafish Plk1 (1) as well as hydrogen bonds (2) and hydrophobic packing (3) between NLS2 and the PBD. Residues participating at the interface are drawn in sticks and are labeled. Equivalent residues in Drosophila Polo that were mutated in this study (Fig. 3) are in parentheses. Hydrogen bonds are indicated by red dashed lines. Intervening water molecules are indicated by a cross. Structural rendering was performed using PyMOL 1.4 built-in commands
