Abstract
SummaryThe γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a “closed” conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an “open,” nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.
Highlights
Microtubule nucleation in cells is spatially and temporally controlled to ensure proper cytoskeleton function
We find that g-tubulin ring complex (gTuRC) stably caps the minus ends of microtubules that it nucleates stochastically
Filamentous fungi, and metazoans, some GCP2s and GCP3s are replaced in the complex by additional GCP4, GCP5, and GCP6 proteins and in metazoans gTuRC is a stable complex whose assembly is independent of the recruitment to target structures, such as centrosomes (Farache et al, 2018; Lin et al, 2015; Murphy et al, 2001; Oegema et al, 1999; Tovey and Conduit, 2018)
Summary
Microtubule nucleation in cells is spatially and temporally controlled to ensure proper cytoskeleton function. Several proteins have been implicated in activating gTuRC, among which are MZT1 and MZT2, sometimes classified as core components of the metazoan complex (Hutchins et al, 2010; Kollman et al, 2011; Lin et al, 2016; Teixido -Travesa et al, 2012), the recruitment factors CDK5Rap (functional homolog of budding yeast Spc110) (Choi et al, 2010; Lin et al, 2014; Muroyama et al, 2016) and NEDD1 (Scrofani et al, 2015), or microtubule dynamics regulators, such as the microtubule polymerase chTOG (XMAP215) or the multifunctional, catastrophe-suppressing protein TPX2 (Alfaro-Aco et al, 2017; Popov et al, 2002; Scrofani et al, 2015; Thawani et al, 2018)
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