Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

Abstract

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term ‘bridging fibre', bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

PRC 1‐labeled microtubule bundles and kinetochore pairs show one‐to‐one association in metaphase

“…We interpret I b as the signal of the bridging fiber, and I bk as the signal of the bundle consisting of the bridging fiber and the k‐fiber together. In cells treated with control siRNA (control cells), the ratio I b / I bk was 0.44 ± 0.01 ( n = 16 bridges in nine cells, see Table 2 for absolute values of I b and I bk ), consistent with previous results 23. PRC1 knockdown reduced the ratio I b / I bk to 0.33 ± 0.01 ( n = 21 bridges in six cells, P = 0.0003, Fig 4C and Table 2).…”

“…To answer this question, we used only horizontal spindles because spindle length and width could not have been precisely measured in vertically oriented spindles due to the variable tilt of the spindle long axis. We measured the average spindle length to be 10.65 ± 0.16 μm, and the width 11.21 ± 0.25 μm ( n = 29), consistent with previous measurements 23, 34. Both spindle length (Fig 1E ) and width (Fig 1F) increased with the number of coupled kinetochore pairs and PRC1‐labeled fibers.…”

“…A PRC1‐labeled bundle and a kinetochore pair were defined as associated if the distance between them was smaller than 0.3 μm (see Materials and Methods), based on a previous measurement of this distance for outermost kinetochores 23. We found that > 90% of PRC1‐labeled fibers were associated with a kinetochore pair, both in horizontal and vertical spindles ( n > 1,000 PRC1‐labeled bundles in each approach, Fig 1D).…”

“…Previous experiments showed that knockdown of PRC1 by small interfering RNA (siRNA) reduces the thickness of the bridging fiber on the outermost part of the spindle 23. To characterize the effect of PRC1 silencing on the whole metaphase spindle, we used mild PRC1 knockdown in order to maintain spindle integrity in HeLa cells expressing tubulin‐GFP and mRFP‐CENP‐B (Materials and Methods).…”

“…Following the method described in 23, we measured the signal intensity of tubulin‐GFP between sister kinetochores, I b , and across the k‐fiber, laterally of kinetochore, I bk . We interpret I b as the signal of the bridging fiber, and I bk as the signal of the bundle consisting of the bridging fiber and the k‐fiber together.…”