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Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes

Summary During cell division, mitotic spindle microtubules segregate chromosomes by exerting forces on kinetochores. What forces drive chromosome segregation in anaphase remains a central question. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles. Both processes require kinetochores to be linked with the poles. Here we show, by combining laser ablation, photoactivation, and theoretical modeling, that kinetochores can separate without any attachment to one spindle pole. This separation requires the bridging fiber, a microtubule bundle that connects sister kinetochore fibers. Bridging fiber microtubules in intact spindles slide apart with kinetochore fibers, indicating strong crosslinks between them. We conclude that sliding of microtubules within the bridging fibers drives pole separation and pushes kinetochore fibers poleward by the friction of passive crosslinks between these fibers. Thus, sliding within the bridging fiber works together with the shortening of kinetochore fibers to segregate chromosomes.

(A) Time-lapse images of the spindle (top row) in a U2OS cell expressing centromere protein CENP-A-GFP (magenta) and mCherry-α-tubulin (green), and enlargements of the boxed region (middle row). Schemes are shown under the images. K-fiber was cut 2.5 µm from the kinetochore in metaphase (yellow lightning sign). Time 0 is anaphase start. (B) Kymograph (consecutive maximum intensity projections onto the y axis) of the spindle from (A) showing merged channels (top), and traces of displaced kinetochores in magenta and control kinetochores in grey (bottom). (C) Time-lapse images of the spindle (top row) in a U2OS cell, and enlargements of the boxed region (bottom row). After the k-fiber cut 2.5 µm from the kinetochore (yellow lightning sign) in anaphase, the pair of sister kinetochores did not show displacement similar to that in metaphase (compare with Figures 1A and S1A). (D) Distance between spindle pole on the side of the ablation and detached kinetochore (see scheme, n=24 cells) over time, where time 0 is anaphase onset. Individual cells (thin lines), mean (thick line), s.d. (pink region). Note that the detached kinetochores are moving away from the spindle pole. Compare the movement of displaced kinetochores during anaphase with the movement of the control kinetochores in Figure S3A. (E) mCherry-α-tubulin signal intensity of bridging fiber, Ib (blue, measured along blue line on scheme), and the bundle consisting of bridging and k-fiber, Ibk (orange, measured along orange line), after cut in metaphase (left, n=13 cells) and during anaphase (right, n=13 cells). Horizontal lines mark background signal, vertical lines delimit area (grey) where signal was measured.
(F) mCherry-α-tubulin signal intensity of the bundle consisting of bridging fiber and k-fiber, I b /I bk , in a displaced element in anaphase, as a function of I b /I bk in metaphase after the cut (n=13 cells).
(G) Left panel: image of the fixed spindle after ablation (yellow lightning sign) 2.5 um from kinetochore (top row) in a U2OS cell stably expressing centromere protein CENP-A-GFP (magenta) and immunostained for α-tubulin (Alexa Fluor-555 shown in green), and smoothed enlargements of the boxed region in both channels (middle row) and only green channel (bottom row). Right panel: image of the spindle after ablation (yellow lightning sign) 2.5 um from kinetochore (top row) in a U2OS cell stably expressing centromere protein CENP-A-GFP (magenta) and stained with SiR-tubulin (green), and smoothed enlargements of the boxed region in both channels (middle row) and only green channel (bottom row).
(H) Time-lapse images of the spindle (top row) in a U2OS cell expressing centromere protein CENP-A-GFP (magenta) and PRC1-mCherry (green), and smoothed enlargements of the boxed region in both channels (middle row) and only green channel (bottom row). K-fiber was cut 2.5 µm from the kinetochore in metaphase (yellow lightning sign). Time 0 is anaphase start.
(I) Time-lapse images of the enlarged spindle region in a U2OS cell expressing centromere protein CENP-A-GFP (magenta) and mCherry-α-tubulin (green). Time 0 is anaphase start. White cross represents the midpoint between two displaced kinetochores at a beginning of anaphase.
(J) Distance between line connecting displaced sister kinetochores and their midpoint (see scheme, n=24 cells) defined at the beginning of anaphase over time, where time 0 is anaphase onset (left panel). Average distance between detached (blue line) and attached (orange line) kinetochore from their midpoint (n=24 cells) over time, where time 0 is anaphase onset for attached displaced kinetochore (right panel). Note the delay in the anaphase onset for the detached displaced kinetochore. Scale bars, 1 µm; time bars, 1 min; KC, kinetochore. To test whether the continuous ablation had a nonspecific impact on kinetochore velocities, we performed continuous ablation between one spindle pole and the cell cortex with the same ablation parameters as in midzone cut experiment. The average kinetochore separation velocities obtained from that experiment were not different from control values ( Figure 2I, Table 1), suggesting that the observed slower kinetochore and pole separation after severing of the midzone region is not a consequence of unspecific effects of ablation, but rather the result of the disruption of bridging fibers.  Figure 3C and S1D. Note that the control kinetochores are moving towards the spindle pole. (B) L stub (n=9 cells) during metaphase, measured as shown in Figure 3A, over time. Time 0 is anaphase onset. Legend as in (A). Note that the length of the stub remained constant during metaphase. The observed stable stub length in metaphase is contrary to some of the previously reported results (Maiato et al., 2004;Sikirzhytski et al., 2014), probably because in our experiments the newly created stub was completely free from the rest of the spindle, whereas this was not the case in the mentioned studies. (C) Time-lapse images of displaced sister kinetochores and the stub in U2OS cell after k-fiber cut 2.5 µm from the kinetochore. The detached kinetochore remained linked to the plus end of the k-fiber stub throughout anaphase which excluded the possibility that motor proteins move the kinetochore along the stub. (D) Photoactivation of the k-fiber stub tip. Spindle after k-fiber ablation (right, yellow lightning sign), smoothed enlargements of the boxed region in the channel showing CENP-A-GFP and PA-GFP-tubulin (green and magenta, top row) and mCherry-α-tubulin channel (green, middle row) after photoactivation of the k-fiber stub at time 0. White arrowhead marks the photoactivated spot. Signal intensities of the PA-GFP-tubulin between the detached kinetochore and the stub tip in the respective frames above (bottom row). Vertical lines mark the signal intensity peaks, arrows show the approaching of the detached kinetochore to the photoactivated spot. Note that the photoactivated stub tip is stable during time while the stub length is decreasing. The observed stub shortening in anaphase is contrary to the previous observation of stable anaphase stubs (Sikirzhytski et al., 2014), yet this discrepancy may be due to a different imaging duration. Scale bars, 1 µm; KC, kinetochore. (E) Photoactivation of the bridging fiber at the start of the anaphase. Spindle after k-fiber ablation (left, yellow lightning sign), smoothed enlargements of the boxed region in the channel showing mCherry-α-tubulin (green) and PA-GFP-tubulin (magenta) after photoactivation of the bridging fiber at time 0. White arrowheads mark the photoactivated spot, which splits into two spots.  Figure 5B right. Other parameters are as in Figure 5B right; " ± = ±2.5 , "+ ± = ±4 , -. ± = ±2 and / ± = ±6 . (B) and (C) Above, scheme of the model. Below, positions of kinetochores, k-fibers, bridging fibers, and spindle poles are shown as a function of time for the parameters given in Figure 5B. Solid and dashed lines correspond to the upper and lower fiber respectively. (B) Solutions of the modified model in which the forces at the pole and also between the antiparallel MTs in the upper right fibers are set to zero at time = 0.5 min. (C) Solutions of the modified model without the connection between k-fibers and bridging fibers. (D) L stub (see scheme, n=8 cells) over time, where time 0 is anaphase onset for cells containing the data in 0-180 s time interval. Individual kinetochores (colored lines), mean (black line), s.d. (shaded region). (E) Distance between detached kinetochore (KC) and the midpoint (see scheme) over time, where time 0 is anaphase onset, for the same cells as in (D). Legend as in (D).