Related to Fig. 1. (A) Two proposed models of error correction mediated by Aurora B kinase. State 1 is the initial incorrect state. In the release model, microtubules detach from kinetochores (state 2) to allow new attachments to form. The process is iterative until bi-oriented attachments (state 3) are stabilized. In the depolymerization model, microtubules depolymerize while maintaining kinetochore attachment, generating poleward chromosome movement. Microtubules detach near the pole (state 2), followed by translocation along another K-fiber to the spindle equator (state 3), and binding of a microtubule from the opposite pole to achieve bi-orientation (state 4). (B) The photocaged and uncaged chemical dimerizers used in this study. CTH consists of the following components: A coumarin photocage; trimethoprim (TMP), which binds eDHFR; and a Halo ligand, which binds covalently to the HaloTag protein. TNH, consisting of TMP linked to NVOC (6-nitroveratryl oxycarbonyl) and a Halo ligand, was used for dimerization without requiring uncaging. (C) Representative images of the negative control (Video 2; yellow triangles label spindle poles). (D and E) Hec1-Ser44 phosphorylation after 2×INBox recruitment with TNH. Images (D) show immunostaining with a phospho-specific antibody in the presence or absence of 2×INBox recruitment to kinetochores. Quantification of Hec1-S44p signals shows higher phosphorylation with 2×INBox recruitment (n = 10 and 11 cells for nonrecruited and recruited kinetochores, respectively). Black lines: Mean ± SEM. (F–J) Depolymerization depends on Aurora B kinase activity and the location of the recruitment site. Representative images show INBox (F; n = 27) or INBoxAAA (G; n = 15) recruitment to the outer kinetochore anchor SPC25, or INBox recruitment to the inner centromere anchor CENP-B (I; n = 24), using TNH. INBoxAAA denotes alanine substitutions at the INBox TSS motif to prevent kinase activation. Images are maximum-intensity projection across z-slices that cover spindle poles for analyses. Displacements (H and J) quantified as in Fig. 1 G. Black lines: Mean ± SEM. (K–M) Aurora B kinases recruitment to kinetochores on monopolar spindles, using CTH, partially triggers microtubule release (same data as Fig. 1, E and F, Fig. S1 C, and Videos 2, 3, and 4). (K) Radial distance from the outermost kinetochores to the population median in the 10-min time window (same dataset as Fig. 1 G). Most kinetochores move toward the pole after activation, but kinetochores that release microtubules should be pushed away from the pole, leading to a large deviation (M, magenta triangles). Magenta bars indicate two of 41 control (Void-recruited) cells (4.9%), with at least one kinetochore pushed away. Cells with INBox recruitment contain fewer such kinetochores (L; 11.9%) than with 2×INBox recruitment (M; 23.1%). *, P < 0.05. Scale bars: 5 µm or 1 µm in insets.
Figure S1.

Related to Fig. 1. (A) Two proposed models of error correction mediated by Aurora B kinase. State 1 is the initial incorrect state. In the release model, microtubules detach from kinetochores (state 2) to allow new attachments to form. The process is iterative until bi-oriented attachments (state 3) are stabilized. In the depolymerization model, microtubules depolymerize while maintaining kinetochore attachment, generating poleward chromosome movement. Microtubules detach near the pole (state 2), followed by translocation along another K-fiber to the spindle equator (state 3), and binding of a microtubule from the opposite pole to achieve bi-orientation (state 4). (B) The photocaged and uncaged chemical dimerizers used in this study. CTH consists of the following components: A coumarin photocage; trimethoprim (TMP), which binds eDHFR; and a Halo ligand, which binds covalently to the HaloTag protein. TNH, consisting of TMP linked to NVOC (6-nitroveratryl oxycarbonyl) and a Halo ligand, was used for dimerization without requiring uncaging. (C) Representative images of the negative control (Video 2; yellow triangles label spindle poles). (D and E) Hec1-Ser44 phosphorylation after 2×INBox recruitment with TNH. Images (D) show immunostaining with a phospho-specific antibody in the presence or absence of 2×INBox recruitment to kinetochores. Quantification of Hec1-S44p signals shows higher phosphorylation with 2×INBox recruitment (n = 10 and 11 cells for nonrecruited and recruited kinetochores, respectively). Black lines: Mean ± SEM. (F–J) Depolymerization depends on Aurora B kinase activity and the location of the recruitment site. Representative images show INBox (F; n = 27) or INBoxAAA (G; n = 15) recruitment to the outer kinetochore anchor SPC25, or INBox recruitment to the inner centromere anchor CENP-B (I; n = 24), using TNH. INBoxAAA denotes alanine substitutions at the INBox TSS motif to prevent kinase activation. Images are maximum-intensity projection across z-slices that cover spindle poles for analyses. Displacements (H and J) quantified as in Fig. 1 G. Black lines: Mean ± SEM. (K–M) Aurora B kinases recruitment to kinetochores on monopolar spindles, using CTH, partially triggers microtubule release (same data as Fig. 1, E and F, Fig. S1 C, and Videos 2, 3, and 4). (K) Radial distance from the outermost kinetochores to the population median in the 10-min time window (same dataset as Fig. 1 G). Most kinetochores move toward the pole after activation, but kinetochores that release microtubules should be pushed away from the pole, leading to a large deviation (M, magenta triangles). Magenta bars indicate two of 41 control (Void-recruited) cells (4.9%), with at least one kinetochore pushed away. Cells with INBox recruitment contain fewer such kinetochores (L; 11.9%) than with 2×INBox recruitment (M; 23.1%). *, P < 0.05. Scale bars: 5 µm or 1 µm in insets.

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