Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes

Abstract

11The spindle generates force to segregate chromosomes at cell division. In mammalian 12 cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle 13 anchors kinetochore-fibers in space and time to coordinate chromosome movement. 14 Yet, how it does so remains poorly understood as we lack tools to directly challenge this 15 anchorage. Here, we adapt microneedle manipulation to exert local forces on the 16 spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals that the 17 spindle retains local architecture in its center on the seconds timescale. Upon pulling, 18 sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting 19 chromosome stretching. Further, pulled kinetochore-fibers freely pivot around poles but 20 not around chromosomes, retaining their orientation within 3 µm of chromosomes. This 21 local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. 22Together, these observations indicate short-lived, specialized reinforcement of the 23 kinetochore-fiber in the spindle center. This could help the spindle protect local structure 24 near chromosomes from transient forces while allowing its remodeling over longer 25 timescales, thereby supporting robust chromosome attachments and movements. 26 27 48 spindle. K-fibers make contacts along their length with a dense network of non-49 kinetochore microtubules (non-kMTs) (Mastronarde et al., 1993;McDonald et al., 1992), 50 likely through both motor and non-motor microtubule binding proteins (Elting et al., 51 2017;Kajtez et al., 2016;Vladimirou et al., 2013). We know that the non-kMT network 52 bridges sister k-fibers together (Kajtez et al., 2016;Mastronarde et al., 1993; Witt, Ris & 53 Borisy, 1981), and that it can locally anchor k-fibers and bear load in the spindle's 54 longitudinal (pole-pole) axis (Elting et al., 2017). Yet, how the dynamic spindle 55 mechanically anchors k-fibers in space and time remains poorly mapped and 56 understood. Specifically, we do not know if k-fibers are anchored uniformly along their 57 length, to what structures they are anchored to, over what timescale this anchorage 58 persists before remodeling is allowed, or more broadly how local forces propagate 59 through the spindle's longitudinal and lateral axes. These questions are central to the 60spindle's ability to robustly maintain its structure, respond to force and ultimately move 61 chromosomes. 62We currently lack tools to apply forces with both spatial and temporal control to 63 mammalian spindles. For example, laser ablation, commonly used to alter forces in the 64 spindle, can locally perturb spindle structure, but lacks control over the duration and 65 direction of ensuing force changes. Further, mammalian spindles cannot yet be 66 reconstituted in vitro. To understand how the dynamic spindle robustly anchors k-fibers, 67 and to ultimately map mammalian spindle mechanics to function, we need approaches 68 to apply local and reproducible forces inside cells, with spatiotemp...

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

“…Microneedle manipulation was adapted for use in mammalian spindles for sustained 381 periods of many minutes by optimizing needle dimensions, contact geometry, and 382 speed of motion to minimize cellular damage. Further microneedle manipulation details 383 can be found in (Suresh et al, 2019). 384 385…”

“…We adapted microneedle 93 manipulation to pull on individual k-fibers in mammalian cells ( Fig. 1A) and developed 94 methods to do so gently enough to exert force for several minutes (Suresh et al, 2019). 95…”

“…The microneedle only locally 102 deformed the cell membrane and spindle and remained outside of the cell, allowing 103 precise, local control of where force is applied ( Fig. 1C) (Suresh et al, 2019). Upon 104 careful removal of the microneedle, cells typically entered anaphase (Fig.…”

K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

“…K-fiber reinforcement could come from connections between kinetochore microtubules themselves, or from associations with non-kinetochore microtubules. Electron microscopy, microneedle, and laser ablation experiments all indicate that nonkinetochore microtubules form mechanical connections with kinetochore microtubules within the k-fiber (Nicklas et al , 1982;Hays and Salmon, 1990;McDonald et al , 1992;Kajtez et al , 2016;Elting et al , 2017;Suresh et al , 2020) . While it is now clear that these non-kinetochore-microtubules contribute to local force dissipation across the spindle, they may also play a role in mechanical stabilization of the k-fiber itself.…”

“…Yet, k-fibers must not be too stable, or spindles may be unable to correct attachment errors, which can result in mis-segregated chromosomes (Lampson et al , 2004) . Regulation of microtubule lifetime is not the only way that spindles respond to competing demands: evidence also increasingly shows that the magnitude of force generation in the spindle balances robustness and dynamics in a myriad of ways (Pereira and Maiato, 2012;Elting et al , 2014Elting et al , , 2017Kajtez et al , 2016;Long et al , 2020;Suresh et al , 2020) . Yet, how k-fibers tune their mechanical integrity to maintain spindle structure and support accurate chromosome segregation remains unclear.…”