Figure 1.
Figure 1. Microtubule severance triggers a response that rapidly pulls detached microtubules toward spindle poles. (A) To probe forces that maintain spindle poles, we challenge the spindle steady-state by detaching microtubules from poles using laser ablation. (B–F) Time-lapse live images of GFP–α-tubulin PtK2 cells (phase contrast, blue; GFP–α-tubulin, yellow). Time is in min:s, with frame captured immediately after ablation set to 00:00. Bars, 2 µm. Arrowheads mark minus ends of ablated microtubules. Dotted lines indicate the position of the kinetochore end of the ablated k-fiber (B–D and F) or bundle minus end (E) immediately before ablation. (B) Representative response of metaphase spindle to k-fiber laser ablation (marked by X). After ablation, the centromere initially relaxes, causing the ablated k-fiber stub to move upward (00:00–00:08). During this time, the k-fiber stub also rotates freely and the uncapped (and unstable) microtubule plus ends depolymerize. Then, the k-fiber stub is pulled rapidly poleward, stretching the centromere and dragging the attached chromosome poleward (00:08–02:01). Minus ends are reincorporated into the spindle (rightmost panel), and the chromosome then resumes typical metaphase oscillations (not depicted). The kinetochore whose k-fiber is ablated is marked by an asterisk and its sister by an o. See also Video 1. (C) Representative response of anaphase spindle to k-fiber laser ablation (X). After ablation, the k-fiber stub rotates freely and its attached chromatid moves upward (00:00–00:21). Upon apparent contact with a neighboring microtubule (00:21), the k-fiber stub is pulled poleward faster than typical anaphase chromatid movement (times 00:21–01:15). The kinetochore of the ablated k-fiber (*) is pulled rapidly toward the pole, passing a neighboring control chromosome (o). See also Fig. S2 and Video 2. (D) Representative response of monopolar spindle to k-fiber laser ablation (X). Immediately after ablation, the k-fiber stub rotates freely, but its attached chromosome does not move upward, consistent with a lack of force from a sister half-spindle (00:00–00:07). Then, the k-fiber stub is pulled poleward, dragging the attached chromosome by its kinetochore (*; 00:07–00:35). The kinetochore of an unmanipulated neighboring k-fiber is marked by o. See also Video 3. (E) Representative response of monopolar spindle to non–k-fiber bundle laser ablation (X). Almost immediately after ablation, the severed non–k-fiber bundle is rapidly pulled toward the pole (00:06–00:32). See also Video 4. (F) Representative response of cell to ablation (X) of the newly created microtubule minus end. After a first ablation (00:00), the k-fiber stub (kinetochore marked by asterisk) is pulled poleward (00:00–00:53). A second ablation destroys the k-fiber stub minus end (01:03), and poleward movement temporarily stops (01:03–01:10), suggesting that poleward force generation requires the minus end. A second poleward transport phase follows this pause (01:28–01:46). See also Video 7.

Microtubule severance triggers a response that rapidly pulls detached microtubules toward spindle poles. (A) To probe forces that maintain spindle poles, we challenge the spindle steady-state by detaching microtubules from poles using laser ablation. (B–F) Time-lapse live images of GFP–α-tubulin PtK2 cells (phase contrast, blue; GFP–α-tubulin, yellow). Time is in min:s, with frame captured immediately after ablation set to 00:00. Bars, 2 µm. Arrowheads mark minus ends of ablated microtubules. Dotted lines indicate the position of the kinetochore end of the ablated k-fiber (B–D and F) or bundle minus end (E) immediately before ablation. (B) Representative response of metaphase spindle to k-fiber laser ablation (marked by X). After ablation, the centromere initially relaxes, causing the ablated k-fiber stub to move upward (00:00–00:08). During this time, the k-fiber stub also rotates freely and the uncapped (and unstable) microtubule plus ends depolymerize. Then, the k-fiber stub is pulled rapidly poleward, stretching the centromere and dragging the attached chromosome poleward (00:08–02:01). Minus ends are reincorporated into the spindle (rightmost panel), and the chromosome then resumes typical metaphase oscillations (not depicted). The kinetochore whose k-fiber is ablated is marked by an asterisk and its sister by an o. See also Video 1. (C) Representative response of anaphase spindle to k-fiber laser ablation (X). After ablation, the k-fiber stub rotates freely and its attached chromatid moves upward (00:00–00:21). Upon apparent contact with a neighboring microtubule (00:21), the k-fiber stub is pulled poleward faster than typical anaphase chromatid movement (times 00:21–01:15). The kinetochore of the ablated k-fiber (*) is pulled rapidly toward the pole, passing a neighboring control chromosome (o). See also Fig. S2 and Video 2. (D) Representative response of monopolar spindle to k-fiber laser ablation (X). Immediately after ablation, the k-fiber stub rotates freely, but its attached chromosome does not move upward, consistent with a lack of force from a sister half-spindle (00:00–00:07). Then, the k-fiber stub is pulled poleward, dragging the attached chromosome by its kinetochore (*; 00:07–00:35). The kinetochore of an unmanipulated neighboring k-fiber is marked by o. See also Video 3. (E) Representative response of monopolar spindle to non–k-fiber bundle laser ablation (X). Almost immediately after ablation, the severed non–k-fiber bundle is rapidly pulled toward the pole (00:06–00:32). See also Video 4. (F) Representative response of cell to ablation (X) of the newly created microtubule minus end. After a first ablation (00:00), the k-fiber stub (kinetochore marked by asterisk) is pulled poleward (00:00–00:53). A second ablation destroys the k-fiber stub minus end (01:03), and poleward movement temporarily stops (01:03–01:10), suggesting that poleward force generation requires the minus end. A second poleward transport phase follows this pause (01:28–01:46). See also Video 7.

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