Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation

Abstract

15Phase separation of substrates and effectors is proposed to enhance biological reaction rates and 16 efficiency. TPX2 is an effector of microtubule nucleation in spindles, and functions with the 17 substrate tubulin by an unknown mechanism. Here, we show that TPX2 phase separates into a 18 co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated 19 cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules at the 20 endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions 21 of TPX2 directly demonstrate that TPX2-tubulin co-condensation enhances the efficiency of 22 TPX2-mediated microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/β 23 heterodimer, regulates both co-condensation and activity. Our study demonstrates how regulated 24 phase separation can simultaneously enhance reaction efficiency and spatially coordinate 25 microtubule nucleation, which may facilitate rapid and accurate spindle formation. 26 27 28 45 Gunzelmann et al., 2018; Thawani et al., 2018), as well as the protein complex augmin that directly 46 recruits γ-TuRC along the length of a pre-existing MT (Song et al., 2018). Branching MT 47 2018).

Biochemical reconstitution of branching microtubule nucleation

“…This may 168 be reflective of cell types where TPX2 is not needed for branching microtubule nucleation, such 169 as mitotic Drosophila cells (Verma and Maresca, 2019). Unexpectedly, it is beneficial that TPX2 170 binds to microtubules first, as it recruits more augmin, in addition to its ability to recruit tubulin 171 (King and Petry, 2019). This is similar to a recent observation in Xenopus egg extract where TPX2 172 binds to microtubules first, and augmin cannot bind to microtubules in its absence (Thawani et al, for 20 min at 17,000 g at 4°C, the pellet was resuspended in 15 ml of the initial CSF-XB buffer 230 supplemented with 0.05% NP-40.…”

“…In meiotic Xenopus egg extract, the Ran-51 regulated protein TPX2 is released near chromatin (Gruss et al, 2001), where it stimulates 52 branching microtubule nucleation (Petry et al, 2013), potentially by activating g-TuRC (Alfaro-53 Aco et al, 2017). Recently, TPX2 was also observed to form a co-condensate with tubulin along 54 microtubules, which enhances the kinetic efficiency of branching microtubule nucleation (King 55 and Petry, 2019). In meiotic Xenopus egg extract, TPX2 binds to microtubules before augmin/ g-56 TuRC, followed by the nucleation event (Thawani et al, 2019).…”

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

“…TPX2, a protein involved in chromatin-dependent microtubule nucleation, can also stimulate nucleation in vitro Roostalu et al, 2015;Schatz et al, 2003). Its ability to form phase-separated condensates has recently been reported to promote this activity (King and Petry, 2019). We tested here to which extent TPX2 could stimulate microtubule nucleation from immobilized γTuRC.…”