Abstract
Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilises growing microtubule ends and stimulates microtubule nucleation by stabilising early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homolog) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking TPX2’s interaction with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.
Highlights
Spindle assembly and function require precise control of microtubule nucleation and dynamics
Using a Total Internal Reflection Microscopy (TIRFM)-based in vitro assay (Fig. 1c) we investigated how chTOG fused to monomeric GFP localised to dynamic microtubules and how it affected their growth
Microtubules were grown from immobilised microtubule “seeds”, which were stabilised by the non-hydrolysable GTP analogue GMPCPP, and elongated in the presence of Atto647Nlabelled tubulin and GTP41
Summary
Spindle assembly and function require precise control of microtubule nucleation and dynamics. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilises growing microtubule ends and stimulates microtubule nucleation by stabilising early microtubule nucleation intermediates. Importins control the efficiency of the microtubule nucleation by selectively blocking TPX2’s interaction with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells. The chromatin-dependent pathway drives local stimulation of nucleation around chromosomes[8]. A major Ran pathway target required for chromatin-stimulated and augmin-mediated microtubule nucleation and spindle assembly is TPX2 (Targeting Protein for Xklp2)[17,18,19]. Reduction of TPX2 levels increases aneuploidy and elevates frequency of spontaneous tumour development in mice[21], overexpression causes defects in microtubule organisation[20] and correlates with poor cancer prognosis and high metastasis frequency in humans[22]
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