Figure 3. Lateral attachments generating... | Rockefeller University Press

Figure 3.

$Figure 3. Lateral attachments generating long-lived metaphase-level centromere tension do not satisfy the SAC. (A) Time-lapse imaging (maximum-intensity projection) of representative SAC satisfaction kinetics (EYFP-Mad1) and microtubule attachment (mCherry-tubulin) in a PtK2 cell. K2 (orange circle) sits along the side of a neighboring k-fiber, suggesting a lateral attachment, but remains Mad1 positive while its sister K1 (blue circle) loses Mad1. Bottom images display the analysis depicted in B. t = 0 indicates video start. See also Video 4. (B) Analysis of microtubule geometry comparing the tubulin intensity (integrated line scans) inside (Tubin) and outside (Tubout) the pair in A. The intensity difference is high on K1, indicating an end-on attachment, and is near zero on K2, indicating a lateral attachment. (C) Individual, mean, and SEM of Tubout − Tubin for K1 (blue) and K2 (orange) in pairs where K2 begins Mad1 positive (n = eight pairs). AU, arbitrary unit. (D) Individual, mean, and SEM of K–K distances for the traces in C (n = eight pairs). (E) Individual, mean, and Mad1 intensity for K1 (end-on; blue) and K2 (lateral; orange) in C and D (n = eight pairs). (F) Time-lapse imaging (maximum-intensity projection) of representative SAC inactivation kinetics (EYFP-Mad1) and microtubule attachment (SiR-tubulin) at a kinetochore pair (CenpC-mCherry) in a PtK2 cell where some kinetochores are locked in a CenpE-mediated lateral attachment (90 nM GSK-923295 CenpE inhibitor) for >15 min. (A and F) Bars: 3 µm (top); 1 µm (bottom). t = 0 indicates video start. See also Video 5. (G–I) Analysis (individual, mean, and SEM) of fractional position along the pole-to-pole axis (G; also see Fig. 1 D), Mad1/CenpC intensity ratio (H), and K–K distance for such K2 kinetochores highlighted in F (I), where t = 0 indicates video start (n = 10 kinetochores). Dashed black lines in I indicate reference mean K–K distances (same data as Fig. 2 D) for pairs in nocodazole (bottom; n = 24 pairs) and at metaphase (top; n = 40 pairs).$

Lateral attachments generating long-lived metaphase-level centromere tension do not satisfy the SAC. (A) Time-lapse imaging (maximum-intensity projection) of representative SAC satisfaction kinetics (EYFP-Mad1) and microtubule attachment (mCherry-tubulin) in a PtK2 cell. K2 (orange circle) sits along the side of a neighboring k-fiber, suggesting a lateral attachment, but remains Mad1 positive while its sister K1 (blue circle) loses Mad1. Bottom images display the analysis depicted in B. t = 0 indicates video start. See also Video 4. (B) Analysis of microtubule geometry comparing the tubulin intensity (integrated line scans) inside (Tub_in) and outside (Tub_out) the pair in A. The intensity difference is high on K1, indicating an end-on attachment, and is near zero on K2, indicating a lateral attachment. (C) Individual, mean, and SEM of Tub_out − Tub_in for K1 (blue) and K2 (orange) in pairs where K2 begins Mad1 positive (n = eight pairs). AU, arbitrary unit. (D) Individual, mean, and SEM of K–K distances for the traces in C (n = eight pairs). (E) Individual, mean, and Mad1 intensity for K1 (end-on; blue) and K2 (lateral; orange) in C and D (n = eight pairs). (F) Time-lapse imaging (maximum-intensity projection) of representative SAC inactivation kinetics (EYFP-Mad1) and microtubule attachment (SiR-tubulin) at a kinetochore pair (CenpC-mCherry) in a PtK2 cell where some kinetochores are locked in a CenpE-mediated lateral attachment (90 nM GSK-923295 CenpE inhibitor) for >15 min. (A and F) Bars: 3 µm (top); 1 µm (bottom). t = 0 indicates video start. See also Video 5. (G–I) Analysis (individual, mean, and SEM) of fractional position along the pole-to-pole axis (G; also see Fig. 1 D), Mad1/CenpC intensity ratio (H), and K–K distance for such K2 kinetochores highlighted in F (I), where t = 0 indicates video start (n = 10 kinetochores). Dashed black lines in I indicate reference mean K–K distances (same data as Fig. 2 D) for pairs in nocodazole (bottom; n = 24 pairs) and at metaphase (top; n = 40 pairs).