Haren, L. | bioRxiv Science

bioRxiv2020

Sub-centrosomal mapping identifies augmin-γTuRC as part of a centriole-stabilizing scaffold

Centriole biogenesis and maintenance are crucial for cells to generate cilia and assemble centrosomes that function as microtubule organizing centers (MTOCs). Centriole biogenesis and MTOC function both require the microtubule nucleator {gamma}-tubulin ring complex ({gamma}TuRC). The widely accepted view is that {gamma}TuRC localizes to the pericentriolar material (PCM), where it nucleates microtubules. {gamma}TuRC has also been observed at centriolar regions that lack PCM, but the significance of these findings is unclear. Here we have used expansion microscopy to map spatially and functionally distinct sub-populations of centrosomal {gamma}TuRC including in the centriole lumen. Luminal localization is mediated by augmin and both complexes are linked to the centriole inner scaffold through POC5. Disruption of luminal localization impairs centriole stability and cilia assembly, defects that are also observed in {gamma}TuRC mutant fibroblasts derived from a patient suffering from microcephaly with chorioretinopathy. These results identify a novel, non-canonical role of augmin-{gamma}TuRC in the centriole lumen that is linked to human disease.

cell biology↗

bioRxiv2020

A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

{gamma}-tubulin is a major protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (gamma-tubulin complex proteins): {gamma}-tubulin small complexes ({gamma}TuSCs), containing {gamma}-tubulin and GCPs 2 and 3, and {gamma}-tubulin ring complexes ({gamma}TuRCs), containing multiple {gamma}TuSCs, in addition to GCPs 4, 5, and 6. Whereas the structure and assembly properties of {gamma}TuSCs have been intensively studied, little is known about the assembly of {gamma}TuRCs, and about the specific roles of GCPs 4, 5, and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt-resistant sub-complex within the {gamma}TuRC that assembles independently of the presence of {gamma}TuSCs. Incubation of this sub-complex with cytoplasmic extracts containing {gamma}TuSCs leads to the reconstitution of full-sized {gamma}TuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the amino-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs, and we demonstrate that these are involved in the assembly or stabilization of the {gamma}TuRC. Summary statement{gamma}-tubulin ring complexes are templates for microtubule nucleation, composed of {gamma}-tubulin and GCP proteins. GCPs 4, 5, 6 form a stable sub-complex, driving the assembly of the full complex.

cell biology↗