Figure 2. PLK-1 Is Recruited to the NE in Prophase through Its Polo-Box Domain in Early C. elegans Embryos(A) (a) Schematic of PLK-1::sGFP fusion. PLK-1 N-terminal serine/threonine kinase domain (KD, dark blue) and C-terminal Polo-box domain (PBD) containing twoPolo boxes (PB1 and PB2, orange) are represented. (b) Western blot analysis of embryonic extracts from wild-type and PLK-1::sGFP expressing strains usingPLK-1 (upper panel) and tubulin antibodies (lower panel, loading control). (c) Spinning disk confocal micrographs of early embryos expressing PLK-1::sGFP andmCherry::HIS-11 at the one-cell (P0) and two-cell stages (AB and P1). Insets are higher magnification of the boxed regions. Scale bars, 10 and 5 um.(B) Timing of PLK-1::sGFP recruitment to the NE (arrow) relative to NEBD defined as the time point at which the nuclear envelope starts to deform (arrowhead).Scale bar, 5um.(C) (a) Schematic of GFP::PBD fusion protein. (b) Western blot analysis of embryonic extracts from wild-type and GFP::PBD expressing strains using PLK-1 (upperpanel), GFP (middle), and tubulin (lower panel, loading control) antibodies. (c) Spinning disk confocal micrographs of early embryos expressing GFP::PBD at theone-cell and four-cell stages. Scale bars, 10 and 5 um.(D) Model of the tridimensional structure of the C. elegans PLK-1 PBD. Residues H542 and K544 of the PB2 contacting the phosphopeptides and residue M547that is mutated in the
plk-1(
or683ts) allele are highlighted in green and purple, respectively.(E) SPAT-1 interacts with PLK-1 PBD wild-type but not with its H542A/K544M or M547K mutant forms in a yeast two-hybrid assay. Interactions were determinedby assaying growth of yeast cells on selective medium (-L-W-H+3AT, 25 mM). f, Empty Gal4 AD plasmid. Proper expression of the constructs was validated bywestern blot (Figure S1D, b).(F) Western blot analysis of embryonic extracts from N2 animals (wild-type) exposed to mock RNAi (ctrl), or
plk-1(RNAi), and
plk-1(
or683ts) animals raised at 15Cor shifted 5 hr at 25C using PLK-1 (upper panel) and tubulin (lower panel, loading control) antibodies. Quantification of the ratio of PLK-1 versus tubulin signalintensity revealed that PLK-1 levels were reduced by 2.5 times in
plk-1(
or683ts) embryos shifted 5 hr at 25C compared with the control.(G) Confocal images of fixed wild-type and
plk-1(
or683ts) embryos shifted 5 hr at 25C stained with PLK-1 antibodies (red) and counterstained with DAPI (blue).Insets are higher magnification of the boxed regions. Scale bar, 10 um. The arrows point to the maternal and paternal chromosomes segregating without mergingduring anaphase, resulting in the formation of paired nuclei at the two-cell stage in
plk-1(
or683ts) embryos.See also Figure S1.