Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

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.

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

“…Untagged EB1 was expressed and purified as described (Roostalu et al , 2015). The final protein concentration was 1.2 mg/ml.…”

“…All samples were imaged at 30°C on a TIRF microscope (iMIC, FEI Munich) described in detail previously (Duellberg et al , 2014; Maurer et al , 2014; Roostalu et al , 2015). Image acquisition and channel alignment were carried out as explained previously (Maurer et al , 2014).…”

“…To quantify and compare the fluorescence intensities of GFP‐dynein at microtubule plus ends in end tracking experiments, averaged intensity profiles were generated as described previously (Roostalu et al , 2015). In brief, kymographs of dynamically growing microtubules were generated using ImageJ Multiple Kymograph plug‐in for the GFP‐dynein and fluorescently labelled microtubule channels.…”

Suppression of microtubule assembly kinetics by the mitotic protein TPX2

“…1D) is that TPX2 reduces the effective tubulin concentration in solution by sequestering free tubulin into multi-subunit complexes (Gruss et al, 2002;Roostalu et al, 2015;Scrofani et al, 2015). If this is the case, suppressed tubulin subunit on-rates could be an indirect consequence of enhanced formation of tubulin-TPX2 nucleation complexes (Fygenson et al, 1995;Schek et al, 2007;Scrofani et al, 2015).…”

“…Thus, TPX2 functions in part as a scaffold to bring mitotic regulators to spindle microtubules. Recent in vitro studies have demonstrated that TPX2 can promote the nucleation of microtubules in vitro (Roostalu et al, 2015;Wieczorek et al, 2015); however, the molecular mechanism by which this is accomplished remains incompletely understood.…”