The mitotic spindle assembles from microtubules and motor proteins into a bipolar shape, yet in cell-free Xenopus egg extracts, self-organizing structures mature stably into either bipolar or faulty multipolar architectures through distinct growth paths. Work by Fukuyama, Yan, and colleagues showed that multipolar spindles contain a fast bidirectional microtubule flow between poles, indicative of motor-generated extensile (pole-pushing) forces that oppose contractile forces from dynein and kinesin-14 motors, and that a template-guided nucleation mechanism ('guided polymerization') locks in morphological errors. However, the molecular identity and spatial regulation of the motors generating the fast interpolar flow remain unestablished, direct quantitative measurements of the extensile-versus-contractile force balance at spindle poles are still lacking, and correction of an established multipolar spindle into a bipolar one has not been demonstrated. The problem is to determine the active microtubule mechanics — which motors and crosslinkers act where, with what force magnitudes, and how their balance is tuned — that selects bipolar over multipolar spindle architecture.

Background

The mitotic spindle is the microtubule-based apparatus that segregates chromosomes during cell division; its bipolar shape is essential for faithful segregation. Xenopus egg extracts are a cell-free cytoplasm preparation that recapitulates spindle assembly outside the living cell, allowing hundreds of spindles to be imaged while they self-organize in bulk cytoplasm. Individual microtubules — protein polymers built from tubulin subunits — turn over on the timescale of tens of seconds, whereas spindle assembly takes about an hour, so the mature architecture must be transmitted through continuous replacement of its building blocks. Motor proteins of the kinesin and dynein families walk along microtubules and crosslink neighboring polymers: kinesin-5 (also called Eg5) is a homotetrameric motor that crosslinks antiparallel microtubules (polymers oriented with opposite polarity) and pushes them apart, generating extensile force; dynein and kinesin-14 (the Drosophila member is called Ncd) are minus-end-directed motors that can pull structures together, generating contractile force. Augmin is an octameric protein complex that nucleates new microtubules as branches growing off the sides of existing ones. Fluorescence speckle microscopy visualizes the movement of microtubule polymers inside the spindle by tracking sparse fluorescent tubulin clusters ('speckles').

Studying self-organization in Xenopus extracts with automated shape analysis, Yan and colleagues (preprint, 2020; published in expanded form as Fukuyama et al., PNAS 2022) found that structures maturing into bipolar spindles follow growth trajectories distinct from those ending in multipolar (e.g., tripolar) shapes, and that mature phenotypes rarely interconvert. Speckle microscopy revealed, in tripolar spindles, a fast (10-12 micrometers per minute, versus the 2-4 micrometers per minute of ordinary flow) bidirectional microtubule flow between pairs of poles, implying antiparallel bundles whose motor-driven sliding pushes poles apart and prevents their coalescence — consistent with earlier in vitro measurements of pushing forces generated by ensembles of kinesin-5 (Shimamoto et al., 2015) and with the intrinsic contractility of extract microtubule networks driven by dynein (Foster et al., 2015). The authors proposed that proper control of the magnitude and location of this extensile pole-pushing force, balanced against contractile pulling, is what establishes bipolarity while preventing multipolarity. The published PNAS version strengthened this picture: dual-microneedle (fine glass needle) mechanical splitting of a pole converted bipolar spindles into persistently multipolar ones in a fraction of trials or let them recover, depending on the stretch magnitude and duration; post-deformation speckle imaging showed newly polymerized microtubules following the orientation of pre-existing ones, supporting a 'guided polymerization' mechanism in which continuous nucleation off the existing structure locks in the architecture; and pharmacologic perturbation implicated kinesin-5 and augmin in separating or stabilizing the bipolar and multipolar growth paths. Microneedle manipulation of mammalian spindles has likewise revealed spatially distinct mechanics (Suresh et al., 2020, 2022).

Problem Statement

Determine the active microtubule mechanics that select bipolar versus multipolar architecture during spindle self-organization in Xenopus egg extracts, in the specific sense established by Yan et al. (arXiv:2011.14592) and Fukuyama et al. (PNAS 2022): (i) identify the motors and crosslinkers — and their spatial regulation — that generate the fast bidirectional interpolar microtubule flow observed between spindle poles, and establish how their activities are tuned so that assembly yields a bipolar spindle rather than a locked multipolar state; (ii) establish whether antiparallel interpolar bundles generating extensile forces are present and mechanically active at early assembly phases, and whether such early extensile activity biases the subsequent trajectory toward multipolarity; (iii) quantitatively measure the magnitudes of the competing extensile (candidate kinesin-5) and contractile (candidate dynein/kinesin-14) contributions to pole-pole force interactions, by direct mechanical or molecular force measurement; and (iv) determine whether an established multipolar spindle can be corrected into a bipolar one by mechanical or molecular perturbation of the pre-existing microtubule template, given that the reverse (bipolar-to-multipolar) switching and guided-polymerization locking have already been demonstrated. These four questions jointly support one objective — a causal, quantitative account of the extensile-versus-contractile force balance that controls spindle pole numericity (the number of spindle poles) — and an answer should address them as parts of that account rather than as isolated observations: all four parts are mandatory components of that single account, and resolving any one of them, or any subset, in isolation does not resolve the task.

References

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