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. 2019 Sep 6;218(11):3583–3596. doi: 10.1083/jcb.201902105

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© 2019 Kuhn and Dumont

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Figure 4. — The mammalian SAC does not detect changes in spindle-pulling forces at individual kinetochores. (A) Schematic of different spatial arrangements used to probe the role of tension in the SAC. After biorientation, both centromere and kinetochore are under force (left). Preventing biorientation in monopolar or mitosis with an unreplicated genome spindles removes force (red) across the centromere, but force (red) can still be generated across the kinetochore through polar ejection forces generated by chromokinesins (purple; middle). By removing pulling force using laser ablation (X), force can in principle be generated neither across the centromere nor the kinetochore. (B) Time-lapse imaging of microtubule attachments (EGFP-tubulin) and kinetochores (Hec1-EGFP) in a metaphase PtK2 cell under Hec1 RNAi + partial NuMA RNAi during the mechanical isolation of the highlighted k-fiber (circles) using laser ablation (red X, t = 0). Bottom: Schematic and zoom of highlighted pair. Scale bars = 3 µm (large) and 1 µm (zoom). (C) Mean, SEM, and individual K-K distance of pairs before and after ablation (time of fixation is ∼30 s from the end of trace). Vertical dashed line marks first ablation. Horizontal dashed line marks the average K-K distance in 5 µM nocodazole (n = 30 kinetochores). Example in B is the purple trace. (D) Normalized distance along the pole-to-pole axis for disconnected kinetochores before and after ablation. Dashed line marks first ablation, and X’s indicate ablation position. Example in B is the purple trace. (E) Immunofluorescence imaging (maximum-intensity projection) of microtubule attachment (tubulin), kinetochores (CREST), and SAC activation (Mad2) at (left) the cell in B and (right) a prometaphase cell on the same dish at approximately t = 3:40. Scale bars = 3 µm (large) and 1 µm (zoom). (F–H) Individual (circles) and average (lines) K-K distance (F), k-fiber intensity (G), and SAC activation (H; Mad2/CREST, normalized to prometaphase intensity) at ablated kinetochores (n = 5), same-cell controls without ablation (n = 30), and prometaphase cells on the same dish (n = 20). There is no SAC activation and no change in attachment intensity on sister kinetochores attached to an ablated k-fiber, and ablation reduces K-K distance to a value similar to that in nocodazole (P = 0.40).