Individual mammalian k-fibers switch to persistent lengthening in response to sustained applied force. (A) Assay to locally exert force on an outer k-fiber using a microneedle (yellow circle) to probe its response to force (yellow arrow). Possible outcomes include global movement of the whole spindle and local deformation of the k-fiber, reflecting global and local dissipation of applied force, respectively. vs., versus. (B) Representative time-lapse images of spindle and k-fiber (SiR-tubulin, white) movement and remodeling in response to sustained force from a microneedle (Alexa 555, yellow) as in Fig. 1 B. The whole spindle rotates and translates while the k-fiber proximal to the microneedle (white line, tracked) bends and lengthens compared with a control k-fiber (red line, tracked). Scale bar, 4 µm. See also Video 2. (C) Maps of the tracked k-fiber shapes and positions for control and manipulated k-fibers from B. Open circles indicate plus-end positions and filled circles indicate pole positions. The manipulated k-fiber (right) translates in the xy-plane and bends and lengthens over time; the control k-fiber (left) similarly translates, but does not lengthen. (D) Speed of proximal pole (left) and plus end (kinetochore, right) movement relative to the speed of microneedle movement within a half-spindle. Half-spindle movement is positively correlated with microneedle speed, indicating global dissipation of force (pole: Spearman R = 0.48, P = 0.04; plus end: Spearman R = 0.71, P = 0.0007; n = 18 cells). (E) The k-fiber length as a function of time, normalized by subtracting the initial length at start of force application (t = 0) for k-fibers manipulated (right, black; n = 18 cells), in the middle of the half-spindle (middle, blue; n = 13 cells), and on the opposite side of the half-spindle (left, red; n = 18 cells). The micromanipulated k-fiber lengthens persistently during force application while the other k-fibers grow and shrink, but do not systematically change length. (F) Average k-fiber lengths at start and end of force application as a function of k-fiber position in the half-spindle. The manipulated (Manip.) k-fiber (black, n = 18 cells) significantly increased in length (P = 0.0002, two-sided Wilcoxon signed-rank test), while the middle and outer k-fiber lengths remain unchanged (P = 0.73, n = 13 cells and P = 0.35, n = 18 cells, two-sided Wilcoxon signed-rank test). Plot shows mean ± SEM. n.s., not significant. (G) Plot of average k-fiber growth rate for manipulated (Manip.) k-fibers (black, n = 18 cells) compared with middle k-fibers (blue, n = 13 cells) or outer k-fibers (red, n = 18 cells) in the same half-spindle. Only the manipulated k-fiber lengthened significantly during force application while neighboring k-fibers continued oscillating between lengthening and shortening phases (manipulated k-fiber versus middle k-fiber “net,” P = 1.6 × 10−5; manipulated k-fiber versus outer k-fiber net, P = 1.4 × 10−5× 10−5; middle k-fiber net compared with outer k-fiber, P = 0.3, two-sided Mann–Whitney U test). The growth rate of the manipulated k-fiber was not significantly different from the growth rate of the middle k-fiber during just the growth phases of its oscillations (blue ‘growth’, P = 0.98, two-sided Mann–Whitney U test). Plot shows mean ± SEM. n.s., not significant. (H) Growth rate of the manipulated k-fiber as a function of the speed of microneedle movement. The growth rate of the manipulated k-fiber did not correlate with the speed of microneedle movement (Spearman R = −.01, P = 0.98, n = 18 cells). (I) Growth rate of the manipulated k-fiber as a function of distance between the microneedle center and the k-fiber plus end. The growth rate of the manipulated k-fiber does not correlate with the proximity of the microneedle to the plus end (Spearman R = 0.08, P = 0.76, n = 18 cells).
