Persistent Plk1 activity at kinetochores disrupts both interkinetochore tension and intrakinetochore stretch. (A) Schematic of Hec1 and Hec1-Plk1^T210D constructs. (B) The YFP/TFP emission ratio was analyzed in cells expressing a kinetochore-targeted Plk1 phosphorylation sensor, together with either Hec1, Hec1-Plk1^T210D, or Hec1-Plk1^K82R (kinase-inactive mutant), under the conditions indicated (n ≥ 10 cells, n ≥ 15 kinetochores per cell). (C–E) Cells expressing either Hec1 or Hec1-Plk1^T210D were imaged live after nocodazole washout. Images (C) are maximal intensity projections of confocal z series. Insets are optical sections showing individual kinetochore pairs. Note that Hec1-Plk1^T210D localizes to both kinetochores and spindle poles. Metaphase alignment (D) and interkinetochore distance (E) were calculated at each time point (n ≥ 40 kinetochores per time point from multiple cells). (F–I) Cells expressing CENP-T–GFP, together with either Hec1 or Hec1-Plk1^T210D, were imaged live at metaphase. Images (F and G) are single confocal planes, and insets show individual kinetochore pairs used for the line scans. Dashed lines indicate estimated Hec1 and CENP-T positions. Distances were calculated between sister kinetochores (H) or between Hec1 and CENP-T within a kinetochore (n ≥ 80 kinetochore pairs from multiple cells; I). AU, arbitrary unit. (C, F, and G) Bars, 5 µm.