10779/crick.12482189.v1
Tanja Consolati
Tanja
Consolati
Julia Locke
Julia
Locke
Johanna Roostalu
Johanna
Roostalu
Zhuo Angel Chen
Zhuo Angel
Chen
Julian Gannon
Julian
Gannon
Jayant Asthana
Jayant
Asthana
Wei Ming Lim
Wei Ming
Lim
Fabrizio Martino
Fabrizio
Martino
Milos A Cvetkovic
Milos A
Cvetkovic
Juri Rappsilber
Juri
Rappsilber
Alessandro Costa
Alessandro
Costa
Thomas Surrey
Thomas
Surrey
Microtubule nucleation properties of single human γTuRCs explained by their cryo-EM structure.
The Francis Crick Institute
2020
CLMS
MZT2
TIRF microscopy
TPX2
actin
chTOG
cryo-electron microscopy
microtubule nucleation
γ-tubulin ring complex
γTuRC structure
Costa FC001065
SB-ack
EM-ack
CS-ack
FC-ack
PRT-ack
Surrey FC001163
Developmental Biology
06 Biological Sciences
11 Medical and Health Sciences
2020-06-25 14:11:58
Journal contribution
https://crick.figshare.com/articles/journal_contribution/Microtubule_nucleation_properties_of_single_human_TuRCs_explained_by_their_cryo-EM_structure_/12482189
The 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.