KNL1 N terminus is required for Bub1 kinase activity at the kinetochore. (A) Control and Bub1-depleted HeLa cells were immunostained with antibodies to Aurora B pT232 (left panels) or Hec1 pSer44 (right panels). Kinetochore fluorescence intensities are shown. Error bars represent SD from independent experiments (n = 2). For each experiment n ≥ 50 kinetochores were measured from at least 6 cells. (B–D) Flp-In T-REx HeLa cells were depleted of endogenous KNL1, rescued with the indicated GFP-KNL1 fragment upon doxycycline addition, and immunostained with Bub1 (B), histone H2A pT120 (D), or overexpressing hBub1-mCherry (C). Kinetochore fluorescence intensities were quantified. In B and D, error bars represent SD from independent experiments (n = 3 in B; n = 2 in D). For each experiment n ≥ 100 kinetochores, n ≥ 10 cells in B; n ≥ 50 kinetochores, n ≥ 5 cells in D. ***, P < 0.001; NS, not statistically significantly different (Student’s t test). (C) Live-cell imaging in control and 300N KNL1 cells expressing Bub1-mCherry and treated with nocodazole. Time is indicated in minutes. Upon KNL1 depletion 90% of cells had no detectable Bub1 at kinetochores, while 10% of cells showed high levels of Bub1, presumably due to incomplete knockdown. (E) Schematic of GFP-KNL1 300N fragments containing mutations in the MELT or KI motifs. The wild-type (top) and mutated (bottom) sequences are shown. (F) HeLa cells depleted of endogenous KNL1 were rescued with N-terminal GFP-tagged 300N, 300N-KI (300N-KI/A), or 300N-MELT (300N-MELT/A) mutants and immunostained for histone H2A pT120. Bars: (cell panels) 5 µm; (kinetochore pair insets) 0.5 µm.