Figure 4.
Interfaces between mammalian k-fibers and the kinetochore and pole are more robust than k-fiber bundles under sustained force. (A) Three example outcomes of force application (yellow arrow and circle) for several minutes are shown: the k-fiber could detach (purple arrowhead) from the kinetochore (second panel, "+"), the k-fiber could detach (purple arrowhead) from the pole (third panel, "-"), or the k-fiber could remain attached at its ends but break (purple arrowheads) in its center (fourth panel; vs., versus). (B) Representative time-lapse images of k-fiber (GFP-tubulin, white) bending, lengthening, and breaking under sustained force. Before the k-fiber breaks, microtubules appear (insets) on the outside of the deformed k-fiber near the area of high curvature next to the microneedle (Alexa 647, yellow). The break creates new microtubule bundle plus ends (purple arrowheads). Scale bar, 4 µm. See also Video 4. (C) Example map of local curvature (k) along a k-fiber bundle during sustained microneedle manipulation. As the k-fiber bends over time, high curvature (dark red) increases near the microneedle and persists for many minutes before breakage occurs (3.5 min). Open circles indicate plus end positions, and filled circles indicate pole positions. (D) Maximum curvature along the k-fiber in the last tracked time point before breakage in cells with breakage events (purple, n = 6 cells) or at the end of the manipulation for cells with no breakage (black; n = 11 cells). Plot shows mean ± SEM (P = 0.37, two-sided Mann–Whitney U test). (E) Cartoon of two different micromanipulation assays that lead to k-fiber breakage: microneedle is moved continuously at 5.2 ± 0.2 µm/min for 3.1 ± 0.3 min (top, purple) and microneedle is moved at 4.5 ± 0.7 µm/min for 1.7 ± 0.2 min and then held in place until breakage (bottom, green). Plot showing no significant difference in the time at breakage in each assay; plot shows mean ± SEM (n = 7 cells and 4 cells, respectively; P = 0.15, two-sided Mann–Whitney U test). (F) Plot of the average time to a splaying event (where newly visible microtubules appear near the area of high curvature) and average time to breakage for the subset of cells in which both events occurred. Splaying events occurred significantly before breakage events (plot shows mean ± SEM, n = 9 cells, P = 0.02, two-sided Wilcoxon signed-rank test). (G) Example time-lapse images of breakage event in which the newly created bundle plus ends (lower purple arrowhead) are highly stable and persist for minutes after breakage. This example cell is the same as the cell shown in Fig. 3 B, but here the cell is displaying the full response, including breakage. See also Video 5.

Interfaces between mammalian k-fibers and the kinetochore and pole are more robust than k-fiber bundles under sustained force. (A) Three example outcomes of force application (yellow arrow and circle) for several minutes are shown: the k-fiber could detach (purple arrowhead) from the kinetochore (second panel, "+"), the k-fiber could detach (purple arrowhead) from the pole (third panel, "-"), or the k-fiber could remain attached at its ends but break (purple arrowheads) in its center (fourth panel; vs., versus). (B) Representative time-lapse images of k-fiber (GFP-tubulin, white) bending, lengthening, and breaking under sustained force. Before the k-fiber breaks, microtubules appear (insets) on the outside of the deformed k-fiber near the area of high curvature next to the microneedle (Alexa 647, yellow). The break creates new microtubule bundle plus ends (purple arrowheads). Scale bar, 4 µm. See also Video 4. (C) Example map of local curvature (k) along a k-fiber bundle during sustained microneedle manipulation. As the k-fiber bends over time, high curvature (dark red) increases near the microneedle and persists for many minutes before breakage occurs (3.5 min). Open circles indicate plus end positions, and filled circles indicate pole positions. (D) Maximum curvature along the k-fiber in the last tracked time point before breakage in cells with breakage events (purple, n = 6 cells) or at the end of the manipulation for cells with no breakage (black; n = 11 cells). Plot shows mean ± SEM (P = 0.37, two-sided Mann–Whitney U test). (E) Cartoon of two different micromanipulation assays that lead to k-fiber breakage: microneedle is moved continuously at 5.2 ± 0.2 µm/min for 3.1 ± 0.3 min (top, purple) and microneedle is moved at 4.5 ± 0.7 µm/min for 1.7 ± 0.2 min and then held in place until breakage (bottom, green). Plot showing no significant difference in the time at breakage in each assay; plot shows mean ± SEM (n = 7 cells and 4 cells, respectively; P = 0.15, two-sided Mann–Whitney U test). (F) Plot of the average time to a splaying event (where newly visible microtubules appear near the area of high curvature) and average time to breakage for the subset of cells in which both events occurred. Splaying events occurred significantly before breakage events (plot shows mean ± SEM, n = 9 cells, P = 0.02, two-sided Wilcoxon signed-rank test). (G) Example time-lapse images of breakage event in which the newly created bundle plus ends (lower purple arrowhead) are highly stable and persist for minutes after breakage. This example cell is the same as the cell shown in Fig. 3 B, but here the cell is displaying the full response, including breakage. See also Video 5.

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