Mitotic Spindle Assembly: The Roles of Microtubule Nucleation and Assembly

Abstract

In all eukaryotes, the assembly of a bipolar mitotic spindle requires highly ordered arrangement of microtubule arrays. This process is facilitated by the formation of new microtubules and involves many proteins that regulate microtubule organisation and dynamics. While the γ‐tubulin ring complex (γTuRC) determines the spatial and temporal control over the initiation of microtubule growth, recent work reveals the molecular mechanisms behind these dynamic events.

Key Concepts

  • Mitotic spindle is a macromolecular temporary machine that initially assembles in early mitosis and disappears at the end of mitosis.
  • Mitotic spindle mainly consists of microtubules with a variety of microtubule‐associated proteins (MAPs) and motor proteins.
  • Microtubules are assembled from α/β‐tubulin heterodimers that contact both in longitudinal and lateral directions.
  • The MAPs and motor proteins take responsibility for regulating the growth or shrinkage of the microtubules.
  • Microtubule nucleation occurs at microtubule‐organising centres (MTOCs).
  • γ‐Tubulin and several other components assemble into γ‐tubulin ring complex (γTuRC) that determines when and where to nucleate the microtubule.
  • Diffusible RanGTP gradient from chromosomes facilitates chromosome‐mediated microtubule nucleation and assembly.
  • Microtubules may serve as nucleation sites for microtubule nucleation and assembly regulated by γTuRC and augmin protein complex.
  • Small microtubule seed/aster formation and sorting facilitate kinetochore capture by microtubules and mitotic spindle assembly.
  • Mitotic kinases such as cyclin‐dependent kinases, auroras and Plks regulate microtubule nucleation and assembly during mitotic spindle formation.

Keywords: mitotic spindle assembly; microtubule nucleation; microtubule assembly; γTuRC; mitotic kinase; microtubule‐associated proteins (MAPs); motor proteins; Ran GTPase; augmin

Figure 1. Microtubule dynamics during mitotic spindle assembly. (a) Mitotic spindle mainly consists of microtubules whose intrinsic polarity and dynamic growth and shrinkage are critical for the mitotic spindle assembly. (b,c) Growing microtubule end shows flattened structure covered with GTP‐tubulin cap, while shrinking microtubule end, which lacks GTP‐tubulin cap, shows unconsolidated structure. The switch from growing to shrinking is termed ‘catastrophe’, and the conversion from shrinkage to growth is called ‘rescue’. The conformation change of these two states is triggered by GTP hydrolysis. There are a variety of microtubule‐associated proteins (MAPs) binding to the ends of the microtubules and regulate their growth or shrinkage. Generally, some MAPs, like XMAP215, tau and doublecortin, serve as microtubule polymerases to help elongation of the microtubules. Oppositely, some other proteins, such as kinesin‐8, kinesin‐13 and kinesin‐14, serve as microtubule depolymerases to accelerate microtubule shrinkage.
Figure 2. Centrosome‐mediated microtubule nucleation. (a) Centrosome is the largest microtubule‐organising centre (MTOC) in cells. Microtubule nucleation relies on a large protein complex, termed γTuRC. The recruitment of γTuRC onto the centrosomes relies on a series of pericentriolar material (PCM) proteins, such as pericentrin, NEDD1, CEP215 and CEP192. (b) Mitotic kinase PLK1 is involved in regulating the centrosome maturation by phosphorylating NEDD1, pericentrin and Nlp to promote or inhibit their functions. Aurora A, another essential kinase for the centrosome maturation, phosphorylates and activates PLK1. (c) Activation of Aurora A needs diverse cofactors including Ajuba, TPX2, CEP192, Bora, Arpc1b, HEF1 and PAK. These cofactors activate the kinase activity of Aurora A by promoting the autophosphorylation of Aurora A at Thr288. B23/nucleophosmin (NPM) triggers Aurora A kinase activation through inducing phosphorylation of Aurora A on Ser89. Moreover, Aurora A functions in promoting the centrosome maturation through phosphorylating its substrates, such as TACC3 and PLK1.
Figure 3. The role of the GTPase Ran in chromosome‐mediated microtubule nucleation and spindle assembly. (a) In the absence of centrosomes, chromatin provides a stable environment for microtubule aster formation. Chromosomes generate a diffusible RanGTP gradient that decreases from chromosome to poles, stimulating microtubule polymerisation. Motor proteins, Ran and other regulators work coordinately and eventually promote the formation of the bipolar mitotic spindle. (b) Hydrolysis cycle of GTP molecule on Ran and release of spindle assembly factors (SAFs) by Ran‐GTP. Ran functions as a molecular switch that changes its interaction with downstream effectors under GTP‐bound or GDP‐bound state. The switch is regulated by its GTPase‐activating protein RanGAP1 with cofactors RanBP1 and RanBP2 and guanine nucleotide exchange factor RCC1.
Figure 4. Augmin‐dependent microtubule branch formation and bipolar mitotic spindle assembly. (a) The rod‐shaped and direct link of microtubule branch in human cells. Electron microscopy tomography reveals a ∼29‐nm rod‐shaped link between mother and daughter microtubules in human U2OS cells. The daughter microtubule tends to grow at a low branch angle (α < 30°) and the same polarity with the mother microtubule in either rod‐shaped or direct link situation, which promotes the efficient polarity organisation of the newly initiated microtubules. (b) Augmin is required for microtubule‐dependent microtubule nucleation. Augmin determines γTuRC localisation on the spindle microtubules, and depletion of augmin subunits decreases the microtubule intensity in the spindle body. It is suggested that any microtubule can serve as the mother microtubule, no matter it is K‐fibre or polar microtubule.
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Further Reading

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Teixidó‐Travesa N, Roig J and Lüders J (2012) The where, when and how of microtubule nucleation ‐ one ring to rule them all. Journal of Cell Science 125: 4445–4456.

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Xu, Xiaowei, Luo, Jia, Jiang, Qing, and Zhang, Chuanmao(Apr 2016) Mitotic Spindle Assembly: The Roles of Microtubule Nucleation and Assembly. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022518]