Abstract
During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.
Highlights
The microtubule (MT) cytoskeleton is essential for correct intracellular organization, cell division and differentiation
We used high-resolution Cryo-electron microscopy (cryo-EM) to visualize the interaction of the central part of TPX2 (Figure 1A, Figure 1—figure supplements 1 and 2B) (Roostalu et al, 2015) with GMPCPP-MTs
This construct maintains the binding specificity of full-length TPX2, despite its reduced affinity and is amenable to structural studies, because it does not induce MT bundling at the high protein concentration typically required for cryo-EM studies of MT-associated proteins (MAPs), in contrast to full-length TPX2 (Brunet et al, 2004; Schatz et al, 2003)
Summary
The microtubule (MT) cytoskeleton is essential for correct intracellular organization, cell division and differentiation. Among them is TPX2 (Wittmann et al, 1998), a MAP from multicellular eukaryotes that is nuclear during interphase (Neumayer et al, 2012) and associates with spindle MTs after nuclear breakdown during mitosis and meiosis (Garrett et al, 2002; Gruss et al, 2002; Heidebrecht et al, 1997; Neumayer et al, 2014). TPX2 is a multifunctional protein with several mitotic/meiotic activities (Neumayer et al, 2014) Both over- and under-expression of TPX2 perturb MT organization, leading to genomic instability, and mutations in TPX2 are correlated with high metastasis frequency in cancer patients (Aguirre-Portoles et al, 2012; Carter et al, 2006; Gruss et al, 2002; Perez de Castro and Malumbres, 2012). TPX2 is a marker for the diagnosis and prognosis of malignancies (Gruss et al, 2002; Heidebrecht et al, 1997; Neumayer et al, 2014)
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