Evidence that a positive feedback loop drives centrosome maturation in fly embryos

Abstract

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here, we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. Our findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos.

Drosophila Sas-6, Ana2 and Sas-4 self-organise into macromolecular structures that can be used to probe centriole and centrosome assembly

“…The mitotic-PCM scaffolding proteins Spd-2 and Cnn are specifically phosphorylated when they are recruited to centrioles to form the mitotic PCM (Alvarez Rodrigo et al, 2019;Conduit et al, 2014a). To test if these proteins were specifically phosphorylated at SAPs, we purified SAPs from unfertilised eggs and centrosomes from fertilised embryos and found that both the SAPand centrosome-associated fractions of Spd-2 and Cnn were similarly phosphorylated ( Figure 3A).…”

“…The SAPs in both asl 1 and asl B46 eggs recruited Sas-4 at slightly elevated levels compared to WT, even when normalising to the amount of GFP signal in each SAP to compensate for the increased size of the SAPs in the asl B46 eggs ( Figure 7D). Strikingly, however, the asl 1 SAPs recruited essentially no detectable Spd-2 or Cnn-the main components of the mitotic PCM scaffold in flies (Alvarez Rodrigo et al, 2019;Conduit et al, 2014a)-and organised essentially no detectable MTs ( Figure 7C,E,F). This finding supports the previous conclusions that the ability to recruit mitotic PCM and organise astral MTs is essentially abolished in asl 1 mutant tissues (Bonaccorsi et al, 1998;Varmark et al, 2007).…”

“…Spd-2 appears to be essential for mitotic PCM assembly in embryos Several lines of evidence indicate that in Drosophila embryos Asl normally helps to recruit Spd-2 to mitotic centrosomes; Spd-2 then recruits Cnn and Polo, which cooperate with Spd-2 to form a robust PCM scaffold that then recruits other PCM components to the mitotic centrosome (Alvarez Rodrigo et al, 2019;Conduit et al, 2014a;Conduit et al, 2014b). Indeed, SAPs in Spd-2 mutant eggs fail to recruit the PCM components Cnn or -Tubulin ( Figure S3).…”

Ana1 recruits PLK1 to mother centrioles to promote mitotic PCM assembly and centriole elongation

“…In vertebrate systems, Cep192 is required for efficient centrosome maturation (Gomez-Ferreria et al, 2007; Zhu et al, 2008) and is phosphorylated by Aurora A to create PBD-binding sites that recruit PLK1; this promotes the mutual activation of both kinases to promote mitotic PCM assembly (Joukov et al, 2010, 2014). In flies and worms, Spd-2/SPD-2 (the fly/worm homologue of Cep192) is concentrated at centrioles and centrosomes and its phosphorylation also helps recruit PLK1 to the mitotic PCM so that it can phosphorylate Cnn/SPD-5 (Decker et al, 2011; Alvarez-Rodrigo et al, 2019). In fly embryos, Spd-2, Polo and Cnn have been proposed to form a positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole (Conduit et al, 2014b; Alvarez-Rodrigo et al, 2019).…”

“…In flies and worms, Spd-2/SPD-2 (the fly/worm homologue of Cep192) is concentrated at centrioles and centrosomes and its phosphorylation also helps recruit PLK1 to the mitotic PCM so that it can phosphorylate Cnn/SPD-5 (Decker et al, 2011; Alvarez-Rodrigo et al, 2019). In fly embryos, Spd-2, Polo and Cnn have been proposed to form a positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole (Conduit et al, 2014b; Alvarez-Rodrigo et al, 2019). In this scenario, Spd-2 is initially recruited to centrioles and is phosphorylated during mitosis, allowing the Spd-2 to form a scaffold that can recruit other PCM proteins and that fluxes outwards from the mother centriole.…”