We hypothesize that the bridging fiber is linked laterally to a k-fiber in the region away from the kinetochore, whereas these two fibers separate from each other near the kinetochoreKajtez 2016 . Therefore, if a k-fiber is severed close to a kinetochore, this kinetochore will move towards its sister kinetochore because the bridging fiber will detach from the k-fiber stub that remains bound to the kinetochore after the severing, and thus will not be able to balance the tension between sister kinetochores (Fig. 1A). Conversely, if the severing is performed far away from the kinetochore, the bridging fiber will remain connected to the severed k-fiber and continue balancing the interkinetochore tension; thus, the kinetochores will not move significantly towards each other (Fig. 1B)Kajtez 2016 .
We used laser ablation to sever k-fibers at various distances from the kinetochore, from 0.3 µm to more than 3 µm, in human U2OS cells stably expressing CENP-A (a kinetochore protein) tagged with green fluorescent protein (GFP) and tubulin tagged with mCherry. Such severing leaves a k-fiber stub attached to the kinetochore, while the other fragment of the severed k-fiber depolymerizesMaiato 2004 Spurck 1990 . The k-fiber stub moves first away from the spindle axis, due to the release of compressive forcesKajtez 2016 . Subsequently, the stub moves towards the proximal spindle pole and back towards the spindle axisElting 2014 Sikirzhytski 2014 Kajtez 2016 . We identified successful k-fiber severing by observing the movement of sister kinetochores and the attached k-fiber stub away from the spindle axis immediately after the severing, or later their movement towards the pole. We measured the movement of kinetochores between the last image before the severing and the first image taken 3 s after severing (see Methods), in order to study the response of the system to the release of forces due to severing, before new forces start acting on the k-fiber stub pulling it towards the pole. We did not analyze the cells in which reconnection of the stub to the spindle microtubules occurred less than 3 s after severing. In order to measure the length of the stub without interference from neighboring k-fibers, we severed only the outermost k-fibers. Examples of severing close to and far from the kinetochore are shown in Fig. 1C and 1D, and Movies S1 and S2, respectively.
The distance between sister kinetochores before severing was 0.81±0.01 µm (n = 100 cells; all results are given as mean±SEM unless otherwise indicated). We found that the distance between sister kinetochores decreased, on average, by 0.08±0.01 µm 3 s after the severing (Fig. 1E). Afterwards, the average interkinetochore distance slowly increased, due to the movement of the stub towards the proximal spindle pole. These results are in qualitative agreement with previous studiesSikirzhytski 2014 Kajtez 2016 .
To assess the contribution of the movement of each sister kinetochore to the decrease in interkinetochore distance, we measured the movement of each kinetochore with respect to the position of the midpoint between the sister kinetochores before the cut. We found that 3 s after k-fiber severing the kinetochore proximal to the severing spot moved by 0.20±0.02 µm towards the midpoint, while its sister moved by 0.11±0.02 µm away from the midpoint, on average (n = 100 cells; Fig. 1F; note that the difference between these mean values is somewhat larger than the average decrease of the interkinetochore distance given above, due to the fact that the interkinetochore axis rotates slightly after severing). These data show that the relaxation of the interkinetochore distance was caused predominantly by the movement of the kinetochore whose k-fiber was severed.
To test whether the directed movement of kinetochores was a local response to severing of the associated k-fiber, rather than a non-specific response of the whole spindle to laser irradiation, we analyzed the movement of the outermost kinetochores on the side of the spindle opposite from the severing location. We found no significant change in the interkinetochore distance (0.01±0.01 µm; n = 100 cells) and no preferred direction of kinetochore movement on the uncut side of the spindle (Fig. 1G, H). Similarly, we measured no significant change in the interkinetochore distance for kinetochore pairs adjacent to those whose k-fiber was severed (0.01±0.02 µm, n = 16 cells; we analyzed only the cells in which the k-fiber adjacent to the severed k-fiber undoubtedly remained intact and the associated kinetochores could be tracked). These control measurements show that the response to severing was localized to the kinetochores associated with the severed k-fiber, rather than a consequence of non-specific effects of laser ablation.
Next, we analyzed how the relaxation of the interkinetochore distance depends on the length of the k-fiber stub. We found larger relaxations for shorter stubs, i.e., for the cuts closer to the kinetochores (Fig. 1I, J). The average relaxation was 0.10±0.01 µm for stubs shorter than 1 µm, whereas it was only 0.04±0.01 µm for longer stubs.
To test the contribution of bridging fibers to interkinetochore tension in other mammalian cells, we performed equivalent experiments and analysis as above on rat-kangaroo PtK1 cells. The cells expressed Hec1-GFP as a kinetochore marker and were microinjected with X-rhodamine-tubulin (Fig. 1K, L and Movies S3, S4). The distance between sister kinetochores before severing was 1.83±0.09 (n = 33 cells). We found that 3 s after severing of a k-fiber, the interkinetochore distance decreased by 0.31±0.04 µm (Fig. 1M). After severing, both kinetochores moved towards the midpoint (Fig. 1N). However, the kinetochore closer to the severing spot moved by 0.26±0.04 µm, whereas its sister moved by 0.06±0.02 µm (n = 33 cells). Thus, the decrease of the interkinetochore distance was caused mainly by the movement of the kinetochore whose k-fiber was severed, as in U2OS cells. Yet, it is not clear why the sister kinetochore, whose k-fiber was not severed, moved towards the midpoint in PtK1 cells, whereas it moved away from the midpoint in U2OS cells. As in U2OS cells, control measurements in PtK1 cells showed that the kinetochores on the opposite side of the spindle displayed neither a significant change in the interkinetochore distance, nor directed movement following the severing (n = 33 cells; Fig. 1O, P). Importantly, we found that the reduction of the interkinetochore distance was larger in spindles in which the k-fiber stub was shorter (Fig. 1R, S). The average relaxation was 0.46±0.06 µm for stubs shorter than 1 µm, whereas it was only 0.24±0.04 µm for longer stubs.
We have shown that severing of a k-fiber closer to the kinetochore results in a larger relaxation of the interkinetochore tension than severing far from the kinetochore, in human U2OS cells and in rat-kangaroo PtK1 cells. Our results on these two cell lines are in qualitative agreement with each other and with the previous measurements on HeLa cellsKajtez 2016 . Yet, the three cell lines show quantitative differences in the kinetochore behavior. The average interkinetochore distance in intact metaphase spindles is largest in PtK1 cells (~1.8 µm), intermediate in HeLa cells (~1 µm), and smallest in U2OS cells (~0.8 µm). After k-fiber severing, in the spindles with the k-fiber stub smaller than 1 µm, the average relaxation of the interkinetochore distance follows the same pattern: it is largest in PtK1 cells (~0.5 µm), intermediate in HeLa cells (~0.2 µm), and smallest in U2OS cells (~0.1 µm). In all the cell lines, there is a transition in the relaxation of the interkinetochore distance at the stub length of roughly 1 µm (Fig. 1J, S and Fig. 5d fromKajtez 2016 ). This finding suggests that the bridging fiber is linked laterally to the k-fibers in the region more than ~1 µm away from the kinetochore, whereas these fibers are disconnected up to ~1 µm from the kinetochore.