Opposing motors provide mechanical and functional robustness in the human spindle

Abstract

At each cell division, the spindle self-organizes from microtubules and motors. How the spindle's diverse motors, often acting redundantly or in opposition, collectively give rise to its emergent architecture, mechanics, and function is unknown. In human spindles, the motors dynein and Eg5 generate contractile and extensile stress, respectively. Inhibiting dynein or its targeting factor NuMA leads to unfocused, turbulent spindles and inhibiting Eg5 leads to monopoles, yet bipolar spindles form when both are inhibited together. What, then, are the roles of these opposing motors? Here we generate NuMA/dynein- and Eg5-doubly inhibited spindles that not only attain a typical metaphase shape and size, but also undergo anaphase. However, these spindles have reduced microtubule dynamics and are mechanically fragile, fracturing under force. Further, they exhibit lagging chromosomes and dramatic left-handed twist at anaphase. Thus, while these opposing motor activities are not required for the spindle's shape, they are essential to its mechanical and functional robustness. Together, this work suggests a design principle whereby opposing active stresses provide robustness to force-generating cellular structures.

Naegleria’s mitotic spindles are built from unique tubulins and highlight core spindle features

“…For this reason, we favor a model in which secondary bundles function similarly to bridging fibers and contribute to spindle elongation by interactions with primary microtubule bundles rather than direct interactions with chromosomes (Figure 6B). 65 Similar to results from human cell lines, 35,57,58 the microtubule bundles in Naegleria spindles twist. This observation implies that Naegleria's mitotic microtubule bundles are physically connected, a hypothesis that may explain their regular spacing within the spindle.…”

“…The function of spindle chirality in human cells may be a passive mechanical response to spindle forces that decreases the risk of spindle breakage under high load. 58,66 In contrast to the left-handed chirality observed in human cell lines, 35,57,58 the majority of Naegleria spindles are right-handed. When hTERT-RPE1 cells are depleted of components of the key spindle regulator augmin, the spindle twist reverses and becomes right-handed, 58 indicating that the chirality of twist is modulated by microtubule-associated proteins.…”

“…Such twist has so far been documented only in HeLa, U2OS, and hTERT-RPE1 cells, where it is generated through the activity of the spindle kinesins Eg5/kinesin-5 and Kif18A/kinesin-8 and regulated by other microtubule-binding proteins. 35,57,58 To quantify the degree of twist in the Naegleria spindle, we traced individual metaphase bundles (Figure 5A) and measured their curvature and twist by fitting a plane to the points representing the bundle and a circle that lies in this plane to the same points. We then estimated bundle curvature as the inverse of the radius of the fit circle, and the twist as the angle between the plane and the z axis divided by the mean distance of these points from the z axis (Figure 5B).…”

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

“…Unlike metaphase, in anaphase twist is mostly absent in HeLa and RPE1 spindles. A recent study found strong left-handed twist during anaphase after combined Eg5 inhibition and NuMa depletion in RPE1 cells, suggesting that opposing motors are required to prevent twisting in the anaphase spindle (Neahring et al, 2021).…”

“…The experimentally measured three-dimensional shapes of the microtubule bundles, primarily bridging fibers that laterally link sister kinetochore fibers, were used to deduce forces and torques in the spindle by comparison with a theoretical model (Novak et al, 2018). Lefthanded twist was also observed in spindles lacking NuMA and kinesin-5 activity in RPE1 cells during metaphase and anaphase (Neahring et al, 2021). Another organism whose spindles are prominently twisted is a unicellular eukaryote, amoeba Naegleria gruberi.…”