Auto-inhibition of Cnn binding to γ-TuRCs prevents ectopic microtubule nucleation and cell division defects

Abstract

Abstractγ-tubulin-ring-complexes (γ-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within γ-TuRC-tethering proteins, including Drosophila Cnn. Binding promotes microtubule nucleation and is restricted to centrosomes, but the mechanism regulating binding remains unknown. Here we identify an extreme N-terminal “CM1 auto-inhibition” (CAI) domain within the centrosomal isoform of Cnn (Cnn-C) that inhibits γ-TuRC binding. Cnn-C is phosphorylated at centrosomes and we find that phospho-mimicking sites within the CAI domain helps relieve auto-inhibition. In contrast, the testes-specific mitochondrial Cnn-T isoform lacks the CAI domain and can bind strongly to cytosolic γ-TuRCs. Ubiquitously expressing a version of Cnn-C lacking the CAI domain leads to major cell division defects, which appears to be due to ectopic cytosolic microtubule nucleation. We propose that the CAI domain folds back to sterically inhibit the CM1 domain, and that this auto-inhibition is relieved by phosphorylation that occurs specifically at centrosomes.

Centrioles generate two scaffolds with distinct biophysical properties to build mitotic centrosomes

“…Fly handling were performed as previously described (Roberts, 1998). cnn HK21 (Megraw et al, 1999;Vaizel-Ohayon and Schejter, 1999) GFP-Cnn (Tovey et al, 2020); CRISPR Knock-in fast folded GFP Ubq-GFP-TACC (Barros et al, 2005) Ubq-NG-Cnn (Wong et al, 2022) tacc stella592 (Lee et al, 2001) aur 1 (Glover et al, 1995) plp 2172 (Spradling et al, 1999) Spd-2 z35711 (Giansanti et al, 2008b) Plk4 Aa74 (Aydogan et al, 2018) Ubq-RFP-Cnn (Conduit & Raff, 2010) Ubq-GFP-TACC-S863L (Barros et al, 2005) Ubq-NG-TACC Generated in this study Ubq-NG-TACC-S863L Generated in this study tacc 59 (Tang et al, 2020); Gift from Fengwei Yu tacc 74 (Tang et al, 2020); Gift from Fengwei Yu Ubq-Aurora A-GFP (Lucas and Raff, 2007) Ubq-Spd-2-RFP (Conduit et al, 2014c) Ubq-Spd-2-SNAP Generated in this study Asl-NG (Steinacker et al, 2022) Ubq-Spd-2-NG (Wong et al, 2022) Ubq-Msps-GFP (Lee et al, 2001) Ubq-Klp10A-GFP Generated in this study ncd-γ-tubulin-37c-GFP (Hallen et al, 2008) UAS-CHC-GFP (Li et al, 2010) Ubq-GFP-Cnn-ΔLZ (Feng et al, 2017) Ubq-GFP-Cnn-ΔCM2 (Feng et al, 2017) (pSas-6)-mNG (Steinacker et al, 2022) mNeonGreen expression driven by the Sas-6's promoter NG-Sas-6 (Steinacker et al, 2022) Ana2-NG (Steinacker et al, 2022) Spd-NG (Wong et al, 2022) NG-Cnn (Wong et al, 2022) Polo-TRAP-GFP (Buszczak et al, 2007); appears to not be fully functional and is only viable as a heterozygote.…”