Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.21.306845v1?rss=1 Authors: Wieczorek, M., Ti, S.-C., Urnavicius, L., Molloy, K. R., Aher, A., Chait, B. T., Kapoor, T. M. Abstract: The formation of cellular microtubule networks is regulated by the {gamma}-tubulin ring complex ({gamma}-TuRC). This 2.3 MDa assembly of >31 proteins includes {gamma}-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a lumenal bridge. The challenge of reconstituting the {gamma}-TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human {gamma}-TuRC ({gamma}-TuRC-GFP), a ~35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex, {gamma}-TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that {gamma}-TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native {gamma}-TuRCs. Electron microscopy reveals that {gamma}-TuRC-GFP resembles the native {gamma}-TuRC architecture, while {gamma}-TuRCmini-GFP adopts a partial cone shape presenting only 8-10 {gamma}-tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating core in the {gamma}-TuRC. Copy rights belong to original authors. Visit the link for more info