Figure 1. Plk1 signaling at the kinetochore requires binding via its PBD.

(a) Schematic indicating orthogonal control of Plk1 alleles. (b) Schematic of complementation strategy. RPE1 cells expressing GFP-Plk1^as (Plk1^as) were stably infected with an empty Flag vector (Vector), Flag-tagged wild-type Plk1 (Plk1^wt), or Plk1 with a wild-type kinase domain, but crippled Polo-box domain that delocalizes the protein (Plk1^aa and Ch-Plk1^aa). (c-f) Delocalized Plk1 fails to restore metaphase chromosome alignment or anaphase chromosome segregation when GFP-Plk1^as is inhibited with 3-MB-PP1. (c) Graph shows average percentage (± SEM) of pre-anaphase mitotic cells at metaphase with fully aligned chromosomes for each cell line challenged with 500 nM 3-MB-PP1 (n=150 cells/experiment; 3 independent experiments). (d) Representative maximal intensity projection micrographs from (c). Arrowheads indicate misaligned chromosomes. (e) Graph shows average percentage (± SEM) of anaphase cells with fully segregated chromosomes for each cell line challenged with DMSO (yellow bar) or 200 nM 3-MB-PP1 (cyan bars) (n=50 cells/experiment; 4 independent experiments). (f) Representative maximal intensity projection micrographs from (e). Arrowheads indicate lagging chromosomes. *P<.05, ***P<0.0001 by one-way ANOVA or unpaired t-test; NS, not significant (panels c,e). Scale bars, 5 μm (panels d,f).