Tovey, C. A. | bioRxiv Science

bioRxiv2022

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes

Microtubule nucleation is mediated by {gamma}-tubulin ring complexes ({gamma}-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes {gamma}-tubulin small complex ({gamma}-TuSC) association to generate cytosolic {gamma}-TuRCs. Unlike {gamma}-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit {gamma}-tubulin complexes to mitotic centrosomes. Here we show that Spd-2 recruits {gamma}-TuRCs formed via the GCP4/5/4/6 core, but that Cnn can recruit {gamma}-TuSCs directly via its well-conserved CM1 domain, similar to its homologues in budding yeast. When centrosomes fail to recruit {gamma}-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data therefore help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organising centres. They also suggest that the dynamic properties of microtubules are influenced by how they were nucleated.

cell biology↗

bioRxiv2020

Phospho-regulated auto-inhibition of Cnn controls microtubule nucleation during cell division

{gamma}-tubulin ring complexes ({gamma}-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within {gamma}-TuRC-tethering proteins, including Drosophila Cnn. Binding promotes microtubule nucleation and is restricted to centrosomes in dividing cells, but the mechanism regulating binding remains unknown. Here we identify an extreme N-terminal "CM1 auto-inhibition" (CAI) domain found specifically within the centrosomal isoform of Cnn (Cnn-C) that inhibits {gamma}-TuRC binding. Robust binding occurs after removal of the CAI domain or with the addition of phospho-mimetic mutations, suggesting that phosphorylation helps relieve inhibition. We show that regulation of Cnn binding to {gamma}-TuRCs is isoform-specific and that mis-regulation of binding can result in ectopic cytosolic microtubules and major defects during cell division. We also find that human CDK5RAP2 is auto-inhibited from binding {gamma}-TuRCs, suggesting conservation across species. Overall, our results shed light on how and why CM1 domain binding to {gamma}-TuRCs is regulated. Summary (for the online JCB table of contents and alerts)We show that auto-inhibition regulates the binding between microtubule nucleating complexes and proteins that tether them to sites of microtubule nucleation. Failure to properly regulate this binding can lead to ectopic cytosolic microtubule nucleation and major defects during cell division.

cell biology↗