The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

Abstract

Mechanical forces produced by motor proteins and microtubule dynamics within the mitotic spindle are crucial for the movement of chromosomes and their segregation into the emerging daughter cells. In addition to linear forces, rotational forces are present in the spindle, reflected in the left-handed twisted shapes of microtubule bundles that make the spindle chiral. However, the molecular origins of spindle chirality are unknown. Here we show that spindles are most twisted at the beginning of anaphase, and reveal multiple molecular players involved in spindle chirality. Inhibition of Eg5/kinesin-5 in a non-cancer cell line abolished spindle twist and depletion of Kif18A/kinesin-8 resulted in a right-handed twist, implying that these motors regulate twist likely by rotating the microtubules around one another within the antiparallel overlaps of bridging fibers. Depletion of the crosslinker PRC1 resulted in a right-handed twist, indicating that PRC1 may contribute to the twist by constraining free rotation of microtubules. Overexpression of PRC1 abolished twist, possibly due to increased torsional rigidity of the bundles. Depletion of augmin led to a right-handed twist, suggesting that twist depends on the geometry of microtubule nucleation. Round spindles were more twisted than elongated ones, a notion that we directly tested by compressing the spindle along its axis, which resulted in stronger left-handed twist, indicating a correlation between bending moments and twist. We conclude that spindle twist is controlled by multiple molecular mechanisms acting at different locations within the spindle as well as forces, and propose a potential physiological role of twist in promoting passive mechanical response of the spindle to forces during metaphase.

Opposing motors provide mechanical and functional robustness in the human spindle

“…Despite their importance to spindle architecture, when dynein and Eg5 are co-depleted, human spindles form as typical bipoles (Florian and Mayer, 2012;Tanenbaum et al, 2008;van Heesbeen et al, 2014). Similar phenomena have been reported in yeast, Drosophila, Xenopus laevis extract, and pig spindles when the homologous kinesin-5 and the dominant end-clustering motor (dynein or a kinesin-14) are inhibited (Ferenz et al, 2009;Mitchison et al, 2005;Rincon et al, 2017;Saunders and Hoyt, 1992;Sharp et al, 1999).…”

“…We found that the kinesins Kif15 and HSET are both required to establish bipolarity ( Figure 1K; Movie S2). Partially depleting Kif15 in doubly inhibited spindles led to the formation of more monopoles (46%; Figures 1K, S1E, and S1F), consistent with its role in generating extensile stress (Sturgill and Ohi, 2013;Tanenbaum et al, 2009;van Heesbeen et al, 2014;Vanneste et al, 2009). Conversely, depleting HSET caused more spindles to remain turbulent (44%), indicating that it performs contractile minus end clustering.…”

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

“…The spindle in Fig. 1 was imaged as described in Trupini c et al (18). Further examples of imaging both live and fixed cells for purpose of measuring twist can be found in Novak et al (17).…”

“…The direction of the imaging is important for determining handedness of the twist (right-or left-handed twist). Examples of microscope settings for the purpose of imaging spindles for measuring twist can be found in Novak et al (17) and Trupini c et al (18).…”

“…We have recently shown that the spindle in human cells is a chiral object, as bundles follow a left-handed helical path (17,18). Chirality is also present in the spindles of amoeba Naegleria gruberi, though the twist is right-handed (19).…”