Mechanisms underlying spindle assembly and robustness

Abstract

The microtubule-based spindle orchestrates chromosome segregation during cell division. Following more than a century of study, many components and pathways contributing to spindle assembly have been described, but how the spindle robustly assembles remains incompletely understood. This process involves the self-organization of a large number of molecular parts - up to hundreds of thousands in vertebrate cells - whose local interactions give rise to a cellular-scale structure with emergent architecture, mechanics and function. In this Review, we discuss key concepts in our understanding of spindle assembly, focusing on recent advances and the new approaches that enabled them. We describe the pathways that generate the microtubule framework of the spindle by driving microtubule nucleation in a spatially controlled fashion and present recent insights regarding the organization of individual microtubules into structural modules. Finally, we discuss the emergent properties of the spindle that enable robust chromosome segregation.

Fig. 1 ∣. Overview of spindle assembly, from molecular parts to cellular-scale properties.

a, Stages of spindle assembly. During prophase, centrosomes separate to opposite sides of the nucleus and chromosomes become condensed, followed by nuclear envelope breakdown at the end of prophase. In prometaphase, microtubules are nucleated from several sources (see also Fig. 2), and kinetochores begin to capture microtubules. Microtubules that interact with chromosome arms aid in chromosome congression to the middle of the cell. In metaphase, chromosomes align to form the metaphase plate, and bundles of microtubules attached to each kinetochore (termed k-fibres) mature. b, Spatial scales of spindle assembly. Spindle assembly requires formation of microtubules from αβ-tubulin dimers. This process relies on nucleation pathways that target the γ-tubulin ring complex (γ-TuRC) to promote microtubule nucleation in specified locations. Individual spindle microtubules assemble into structural modules through the action of crosslinkers and motor proteins. The spindle is characterized by emergent properties – that is, characteristics of the ensemble that are not exhibited by its component parts – such as its material properties and poleward microtubule flux.

Fig. 2 ∣. Microtubule nucleation during spindle assembly.

a, Spontaneous microtubule nucleation from αβ-tubulin dimers is energetically unfavourable and therefore an inefficient process. Nucleation is facilitated by the universal microtubule nucleation module that comprises the chTOG protein and the γ-tubulin ring complex (γ-TuRC), which mainly consists of γ-tubulin and γ-tubulin complex proteins 2–6 (GCP2–6). γ-TuRC provides a microtubule minus end template for nucleation whereas chTOG recruits αβ-tubulin dimers to speed up microtubule assembly. Microtubule nucleation is localized to specific spindle sites, such as centrosomes (b), existing microtubules (c), kinetochores (d) and centromeres via the chromosomal passenger complex (CPC) (e). Chromosomes play a key role in activating the small GTPase Ran to form RanGTP, which forms a gradient around chromosomes following nuclear envelope breakdown. This gradient spatially activates nucleation on existing microtubules (c) by creating a corresponding gradient of active spindle assembly factors (SAFs). Additionally, RanGTP activates nucleation at kinetochores (d). b, The pericentriolar matrix components pericentrin, centrosomal protein 192 (CEP192) and, possibly, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) localize γ-TuRC to the centrosome. c, In microtubule-mediated (branching) nucleation, the γ-TuRC-interacting complex augmin binds and localizes γ-TuRC and chTOG to the microtubule lattice. In Xenopus laevis egg extract, this mechanism also requires the RanGTP-activated SAF TPX2, which facilitates recruitment of the remaining factors to microtubules. This pathway results in microtubules of conserved polarity relative to the pre-existing microtubule, which are nucleated at a shallow angle (<30°) and often parallel to the mother microtubule. d, γ-TuRC is localized to kinetochores by the nucleoporin (Nup) complex Nup107–160 and Nup-interactor ELYS and activated in a RanGTP-dependent manner. e, Formation and localization of the chromosome passenger complex (CPC) to the inner centromere activates the CPC component Aurora B kinase. Aurora B phosphorylates and inactivates the microtubule destabilizing protein MCAK and the tubulin-sequestering protein stathmin to facilitate microtubule nucleation and polymerization towards kinetochores, independently of RanGTP.

Fig. 3 ∣. Structural modules in the spindle.

Microtubule-associated proteins, including motors, crosslinkers and regulators of microtubule dynamics, organize microtubules into distinct structural modules. a, Kinetochore fibres (k-fibres) are bundles of parallel microtubules with their plus ends bound to the outer kinetochore protein NDC80. K-fibres are crosslinked by microtubule-associated proteins, including the kinesin KIF15, the transforming acidic coiled-coil-containing protein 3 (TACC3)–chTOG–clathrin complex, and HURP. b, Bridging fibres are bundles of antiparallel microtubules that maintain tension on k-fibres and align chromosomes. PRC1 and the kinesin-5 Eg5 bind and crosslink antiparallel microtubules, and PRC1 recruits the plus-end-directed motor MKLP1 to bridging fibres. c, Other non-kinetochore microtubules play important mechanical roles in stabilizing the spindle, anchoring k-fibres, and mediating polar ejection forces via the chromokinesins Kid (KIF22) and KIF4A. d, Astral microtubules extend from spindle poles towards the cell cortex, where they interact with the Gɑ_i–LGN–NuMA complex to position the spindle. Their lengths and numbers are regulated by the depolymerases MCAK and KIF18B, that are recruited to plus ends by plus-end-tracking protein EB1. e, Pole focusing is mediated by motors such as dynein that, together with its regulators (NuMA, dynactin), dwells at minus ends and carries these minus end cargos towards neighbouring minus ends. Black arrows indicate directions of motor stepping; blue arrows labelled ‘F’ indicate direction of force on microtubules.

Fig. 4 ∣. K-fibre formation mechanisms.

a, Kinetochore fibre (k-fibre) formation can occur through two pathways, search-and-capture and chromosome-mediated assembly. In search-and-capture, dynamic centrosome-nucleated microtubules ‘search’ the surrounding cytoplasm for kinetochores as they grow and become stabilized upon kinetochore attachment. In chromosome-mediated assembly, microtubules nucleate via chromosome-mediated and kinetochore-mediated pathways described in Fig. 2. b, A nascent k-fibre typically begins to form when a kinetochore binds to the lateral surface of a microtubule. This laterally bound microtubule undergoes reorientation driven by the plus-end-directed kinesin CENP-E and the microtubule depolymerase MCAK, resulting in stable attachment of microtubule plus ends to the outer kinetochore NDC80 complex. The minus ends of these kinetochore-bound microtubules (KMTs) become bound by the minus-end-binding motor complex NuMA–dynein–dynactin. This minus-end-directed motor complex transports KMTs towards spindle poles (arrows). These nascent k-fibres additionally undergo branching microtubule nucleation, which serves to quickly amplify KMT numbers. c, The mature k-fibre forms both direct and indirect connections with the spindle poles, whereby KMTs of a single k-fibre that are bundled together near the kinetochore gradually splay out towards their minus ends to interact with other microtubules. Ongoing microtubule nucleation, mainly through branching nucleation, maintains the density of the k-fibre.

Fig. 5 ∣. Mechanisms underlying spindle robustness: redundancy, anchorage and dynamics.

a, Partial redundancy between motor proteins makes spindle assembly robust to perturbations and variations in motor activity. KIF15 can compensate for Eg5 inhibition to maintain spindle bipolarity. Similarly, NuMA–dynein–dynactin complexes have dominant roles in pole focusing in human spindles but the pole-focusing activity of HSET can compensate for reduced dynein activity. b, Plus-end-specific and minus-end-specific mechanisms reinforce key connections between the kinetochore fibre (k-fibre), pole, and kinetochore and these can be experimentally dissected by microneedle manipulation. On short (<20 s) timescales, PRC1-mediated crosslinking maintains k-fibre orientation, preventing k-fibres from pivoting around kinetochores. On longer (minutes) timescales, this reinforcement near kinetochores relaxes, allowing k-fibres to pivot around kinetochores. Plus ends undergo persistent polymerization while depolymerization at minus ends ceases, allowing the k-fibre to lengthen in response to external manipulation. These responses allow the k-fibre to locally dissipate force (F), remodelling at the individual k-fibre level to preserve global spindle architecture.

Fig. 6 ∣. Spindle material properties.

a, The responses of the spindle to force are spatially and temporally heterogeneous. Along the short axis, Xenopus extract spindles behave elastically on short (<10 s) and long (>100 s) timescales. On intermediate timescales, corresponding to the time required for chromosome movement, the short axis of the spindle exhibits more viscous behaviour, dissipating energy as it deforms. The long axis of the spindle is also mechanically heterogeneous: it is stiffest near the poles and kinetochores but less stiff in the middle. The pole region exhibits solid-like behaviour, recovering its shape after deformation, whereas the middle and equator regions are more fluid-like. b, Dynein and its cofactors NuMA and dynactin generate contractile stress in vitro and in the spindle, clustering minus ends together. Eg5 mediates extensile stress, giving rise to nematic motifs of aligned, mixed-polarity microtubules. These opposing motor activities must be balanced to give rise to a bipolar spindle: an excess of contractile activity leads to monopolar spindles, and an excess of extensile sliding produces turbulent microtubule networks.

Fig. 7 ∣. Spindle scaling mechanisms.

The scale of the spindle is set by extrinsic and intrinsic mechanisms. a, Extrinsic mechanisms, whereby spindle size scales with cell size, include the limiting components model. This model posits that the size of the spindle is limited by the amount of spindle-building components available. Given that the concentration of the limiting component remains equal irrespective of cell size, a larger cell would contain more of this component and be able to build a larger spindle than a smaller cell. b, Extrinsic scaling can also result from changes in the cellular surface area-to-volume ratio. For example, this occurs if an inhibitor of a negative regulator of spindle size localizes to the cell membrane. As a result, in smaller cells with larger membrane-to-cytoplasm ratios, the inhibitor becomes sequestered at the membrane and enables the negative regulator to scale spindle size down. c, Intrinsic mechanisms for setting maximum spindle length have also been proposed. In very large cells such as early embryonic cells, spindle size can be determined by the scale of a gradient of microtubule nucleation activity such as a RanGTP gradient emanating from the chromosomes.