The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

Abstract

<jats:p>Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 do not enhance γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.</jats:p>

Auto-inhibition of Cnn binding to γ-TuRCs prevents ectopic microtubule nucleation and cell division defects

“…Based on these studies, the CM1 domain has been regarded as an activator of γ-tubulin complexes. Two recent papers, however, reported only a small increase in microtubule nucleation from purified human or Xenopus γ-TuRCs after addition of CM1-domain fragments (Liu et al, 2019; Thawani et al, 2020). Moreover, recent cryo-EM data shows no evidence for structural changes when purified human or Xenopus γ-TuRCs are bound by CM1-domain fragments (Wieczorek et al, 2019; Liu et al, 2019).…”

“…γ-TuRCs contain 14 γ-tubulin molecules held in a single-turn helical shape by laterally associating γ-tubulin complex proteins (GCPs) (Kollman et al, 2011). The γ-tubulin molecules bind directly to a / b -tubulin dimers to promote new microtubule assembly (Thawani et al, 2020). γ-TuRCs have a low activity within the cytosol but are thought to be “activated” after recruitment to MTOCs (Tovey and Conduit, 2018; Farache et al, 2018).…”

“…How activation via an open-to-closed conformation change occurs is currently unclear, but various factors have been reported to increase the capacity for γ-TuRCs to nucleate microtubules. The nucleation capacity of γ-TuRCs purified from Xenopus egg extract is dramatically increased by the addition of the TOG domain protein XMAP215 (Thawani et al, 2020). TOG domain family members mediate a / b -tubulin addition via their TOG domains (Nithianantham et al, 2018), bind directly to γ-tubulin, and function in microtubule nucleation in vivo (Thawani et al, 2018; Flor-Parra et al, 2018; Gunzelmann et al, 2018).…”

“…TOG domain family members mediate a / b -tubulin addition via their TOG domains (Nithianantham et al, 2018), bind directly to γ-tubulin, and function in microtubule nucleation in vivo (Thawani et al, 2018; Flor-Parra et al, 2018; Gunzelmann et al, 2018). Single molecule experiments combined with modelling suggest that XMAP215 indirectly promotes the open-to-closed conformation change of purified γ-TuRCs by increasing the chance of protofilament formation, where the lateral contacts between protofilaments are predicted to force the closure of the γ-TuRC (Thawani et al, 2020). While this is an attractive model, evidence suggests that γ-TuRC activation can occur in different ways and may be context specific.…”

“…Another potential γ-TuRC activator is the Centrosomin Motif 1 (CM1) domain, which is conserved in γ-TuRC-tethering proteins across Eukaryotes (Sawin et al, 2004; Lin et al, 2014b). Addition of protein fragments containing part of the CM1 domain, known as γ-TuNA, dramatically increases the nucleation capacity of γ-TuRCs purified from human cells (Choi et al, 2010; Muroyama et al, 2016), although the degree of this activity change is much lower when using γ-TuRCs purified from Xenopus eggs (Liu et al, 2019; Thawani et al, 2020). While there is evidence that CM1-domain binding does not alter the semi-open conformation of purified γ-TuRCs (Wieczorek et al, 2019; Liu et al, 2019), expression of γ-TuNA within human cells leads to the ectopic nucleation of microtubules throughout the cytosol, and this effect is abolished by the introduction of mutations that prevent CM1 domain binding to γ-TuRCs or by the depletion of γ-TuRC components (Choi et al, 2010; Hanafusa et al, 2015; Cota et al, 2017).…”