Figure 3.
Force on individual mammalian k-fibers suppresses depolymerization at both ends without altering plus-end polymerization rates or inducing microtubule sliding. (A) Assay to determine the physical mechanism of k-fiber lengthening under force by tracking position of a photomark on the k-fiber during microneedle manipulation. Possible outcomes are shown, not mutually exclusive: the photomark could remain fixed relative to the pole, indicating a suppression of minus-end depolymerization (left, blue X); the position of the photomark to the kinetochore could increase continuously, indicating a suppression of plus-end depolymerization or increase in plus-end polymerization rate (middle, blue X or arrow); or the photomark could remain in a fixed position but widen, indicating sliding of microtubules within the k-fiber (right, blue X). (B) Representative time-lapse images of photomark (PA-GFP-tubulin, white) during microneedle (Alexa 555, yellow) manipulation of a k-fiber (SiR-tubulin, magenta). The distance between the photomark and the pole remains constant (orange line), while the distance between the photomark and plus end increases (red line). Scale bar, 4 µm. See also Video 3. (C) Plot of the photomark to pole distance change over time due to flux of microtubules in unmanipulated cells, as a baseline (n = 4 cells). (D) Plot of the photomark to pole distance change during microneedle manipulation, showing that photomark movement poleward due to microtubule depolymerization is suppressed (n = 4 cells). (E) Plot of the photomark to plus end position distance change during microneedle manipulation, showing that k-fibers persistently polymerize at their plus ends under force (n = 4 cells). (F) Representative example of photomark intensity linescans over time during manipulation, from same cell as B. (G) Change in full width at half maximal photomark intensity at each time point during microneedle manipulation, showing that photomarks do not widen under force, and thus that there is no detectable microtubule sliding within the k-fiber (n = 4 cells).

Force on individual mammalian k-fibers suppresses depolymerization at both ends without altering plus-end polymerization rates or inducing microtubule sliding. (A) Assay to determine the physical mechanism of k-fiber lengthening under force by tracking position of a photomark on the k-fiber during microneedle manipulation. Possible outcomes are shown, not mutually exclusive: the photomark could remain fixed relative to the pole, indicating a suppression of minus-end depolymerization (left, blue X); the position of the photomark to the kinetochore could increase continuously, indicating a suppression of plus-end depolymerization or increase in plus-end polymerization rate (middle, blue X or arrow); or the photomark could remain in a fixed position but widen, indicating sliding of microtubules within the k-fiber (right, blue X). (B) Representative time-lapse images of photomark (PA-GFP-tubulin, white) during microneedle (Alexa 555, yellow) manipulation of a k-fiber (SiR-tubulin, magenta). The distance between the photomark and the pole remains constant (orange line), while the distance between the photomark and plus end increases (red line). Scale bar, 4 µm. See also Video 3. (C) Plot of the photomark to pole distance change over time due to flux of microtubules in unmanipulated cells, as a baseline (n = 4 cells). (D) Plot of the photomark to pole distance change during microneedle manipulation, showing that photomark movement poleward due to microtubule depolymerization is suppressed (n = 4 cells). (E) Plot of the photomark to plus end position distance change during microneedle manipulation, showing that k-fibers persistently polymerize at their plus ends under force (n = 4 cells). (F) Representative example of photomark intensity linescans over time during manipulation, from same cell as B. (G) Change in full width at half maximal photomark intensity at each time point during microneedle manipulation, showing that photomarks do not widen under force, and thus that there is no detectable microtubule sliding within the k-fiber (n = 4 cells).

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