Recruitment of Aurora B to syntelic kinetochores triggers microtubule depolymerization. (A) Light-induced dimerization schematic. The dimerizer has a Halo ligand (blue) linked to the eDHFR ligand trimethoprim (TMP; red), which is protected by a photoactivatable cage (purple). The kinetochore protein SPC25 anchors HaloTag at the outer kinetochore, and effectors are fused to eDHFR. Effectors are recruited to kinetochores by uncaging the HaloTag-bound dimerizer with light. (B) Constructs for this study. Dimerizer uncaging recruits eDHFR-tagged effector proteins (or Void as a negative control). The PACT domain targets to centrosomes to label spindle poles (yellow triangles; Gillingham and Munro, 2000). (C) Schematic of monopolar spindle assay, with chromosomes under a tug of war between K-fiber depolymerization and polar ejection forces. Red circles indicate Aurora B–activated kinetochores. After full or partial release of kinetochore microtubules, polar ejection forces dominate and chromosomes move away from spindle poles. In contrast, depolymerization increases poleward forces. (D–F) Representative images before and after uncaging at t = 0 (Videos 1, 2, 3, and 4). (G) Kinetochore displacement over time. For each cell, kinetochore-pole distances are measured and averaged at every time point. Displacements are defined relative to t = 0, with poleward movement defined as the positive direction (Void: n = 41 cells; MCAK: n = 46; INBox: n = 42; 2×INBox: n = 39; mean ± SEM). Scale bars, 5 µm.
Figure 1.

Recruitment of Aurora B to syntelic kinetochores triggers microtubule depolymerization. (A) Light-induced dimerization schematic. The dimerizer has a Halo ligand (blue) linked to the eDHFR ligand trimethoprim (TMP; red), which is protected by a photoactivatable cage (purple). The kinetochore protein SPC25 anchors HaloTag at the outer kinetochore, and effectors are fused to eDHFR. Effectors are recruited to kinetochores by uncaging the HaloTag-bound dimerizer with light. (B) Constructs for this study. Dimerizer uncaging recruits eDHFR-tagged effector proteins (or Void as a negative control). The PACT domain targets to centrosomes to label spindle poles (yellow triangles; Gillingham and Munro, 2000). (C) Schematic of monopolar spindle assay, with chromosomes under a tug of war between K-fiber depolymerization and polar ejection forces. Red circles indicate Aurora B–activated kinetochores. After full or partial release of kinetochore microtubules, polar ejection forces dominate and chromosomes move away from spindle poles. In contrast, depolymerization increases poleward forces. (D–F) Representative images before and after uncaging at t = 0 (Videos 1, 2, 3, and 4). (G) Kinetochore displacement over time. For each cell, kinetochore-pole distances are measured and averaged at every time point. Displacements are defined relative to t = 0, with poleward movement defined as the positive direction (Void: n = 41 cells; MCAK: n = 46; INBox: n = 42; 2×INBox: n = 39; mean ± SEM). Scale bars, 5 µm.

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