Mechanisms of Mitotic Spindle Assembly

Abstract

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.

Keywords: cell division; cytoskeleton; microtubule; microtubule nucleation; microtubule-associated protein; γ-tubulin ring complex.

Figure 1

Anatomy of the metaphase spindle. To date, it has not been possible to directly determine the key parameters that describe microtubule (MT) organization within the spindle, namely the origin, dynamics, polarity, and location of each MT, presumably because the high MT density precludes their direct observation. Markers are lacking for MT minus ends and MT nucleation events, but growing MT plus ends can be tracked via the MT plus-tip tracking protein EB1-GFP (74, 75). Single-molecule speckle imaging has been applied to determine tubulin turnover and MT length distributions in the spindle (78, 79). Lastly, femtosecond laser ablation leads to MT depolymerization of newly generated plus ends, a technique used to quantify MT density and identify the locations of MT plus and minus ends (5). Altogether, these results suggest that MT nucleation combined with local MT transport govern spindle morphogenesis. Abbreviations: EB1-GFP, GFP-labeled end-binding protein 1; K-fiber, kinetochore fiber; KT, kinetochore.

Figure 2

Self-organization of the metaphase spindle. The mitotic spindle is made of up to hundreds of thousands of microtubules (MTs) and roughly 1,000 MT-associated proteins (MAPs), of which ~200 are essential (–25). Spindle MAPs can be grouped into several activity classes, namely MT nucleation, MT dynamics, MT transport, and MT cross-linking. Aside from MT nucleation by the ring-shaped γ-TuRC, the MT number can be increased by MT severing and MT minus-end-binding proteins. MT dynamics are regulated by MT polymerases, MT depolymerases, plus-end tracking proteins (+TIPs), and MT stabilization factors. MT cross-linking can occur at minus and plus ends or along the MT lattice. Finally, molecular motors are responsible for transport on MT tracks. MAPs and MTs self-organize into the functional spindle, whose maintenance continuously consumes and dissipates energy.

Figure 3

Scaling of the metaphase spindle. It is possible to start addressing how spindles assume a certain size and shape. Spindle length can be modulated by varying the concentration and activity of the microtubule (MT) polymerase XMAP215 (81, 82). During later stages of development, the MT depolymerase, Kif2a becomes inhibited and results in smaller spindles (86). Alternatively, the amount of cytoplasmic material can regulate spindle size (87, 88). Differences in the activity of katanin, as well as the activity and concentration of targeting factor for Xklp2 (TPX2), can explain spindle size and architecture changes between large Xenopus laevis and small X. tropicalis spindles (90, 91).

Figure 4

Spindle assembly pathways. The first key step to assemble the mitotic spindle is γ-TuRC–dependent microtubule (MT) (b) nucleation from MT organizing centers. MT nucleation occurs from centrosomes to which γ-TuRC is recruited via its direct binding partners NEDD1 and GCP9 as well as via the centrosomal proteins Cep192 and CDK5RAP2 (, –99, 102, 107, 174). The latter, together with the Aurora A kinase and Plk1, activates MT nucleation and polymerization (e.g., via TACC3/Maskin). (a) Less is known about MT nucleation from acentrosomal poles, which are formed by the combined action of the cross-linker NuMA and dynein (170). Dynein transports γ-TuRC poleward (59). (c) At chromosomes, the CPC inhibits MT depolymerases, such as MCAK, to induce MT formation (59, 72). (d ) The key chromosomal pathway is activated by RanGTP, which in turn releases SAFs from sequestering importins (–131). The central SAF, TPX2, induces MT formation via its N terminus, together with XRHAMM, NEDD1, and γ-TuRC (139). (e) The C terminus of TPX2, together with augmin, induces branching MT nucleation throughout the spindle, a distribution that may be facilitated by the transport of TPX2 by the kinesin Eg5 or a poleward flux (147, 154). ( f ) The SAFs TPX2, HURP, MCRS1, and Nup107–160 play a yet-to-be-defined role in MT nucleation in the vicinity of kinetochores (133, 164, 168). Abbreviations: CPC, chromosomal passenger complex; GCP, γ-tubulin complex protein; γ-TuRC, γ-tubulin ring complex; NEDD1, neural precursor cell expressed, developmentally down-regulated 1; Plk1, Polo-like kinase 1; SAFs, spindle assembly factors; TPX2, targeting factor for Xklp2; XRHAMM, hyaluronan-mediated motility receptor.