Abstract
Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.
Highlights
We previously showed that Cnn and Spd-2 are initially recruited around mother centrioles and spread outward to form an extended pericentriolar material (PCM) scaffold (Conduit et al, 2010, 2014a, 2014b) (Note that we define ‘recruitment’ here as when a new protein molecule is added into the centrosome from the cytosol, irrespective of whether this molecule replaces an existing molecule or adds to the existing pool of molecules)
Cnn has previously been identified as part of a multi-protein ‘S-CAP’ complex, which pre-assembles in the cytosol with the centriole protein Sas-4 before being recruited into the centrosome via a Sas-4–centriole interaction (Gopalakrishnan et al, 2011, 2012; Zheng et al, 2014)
Our results suggest that S-CAP complexes do not play a significant role in mitotic PCM assembly in Drosophila syncytial embryos
Summary
Centrosomes are crucial cell organisers (Nigg and Raff, 2009; Arquint et al, 2014; Chavali et al, 2014; Reina and Gonzalez, 2014; Stinchcombe and Griffiths, 2014). The centrosomal GFP-Cnn and Spd-2-GFP signals recovered immediately, while the Sas-4-mCherry signal only began to recover robustly after the centrosomes separated at the end of mitosis—when a new round of centriole duplication begins (Figure 2; Videos 3, 4; Figure 2—source data 1) These findings are consistent with our hypothesis that Sas-4 molecules are only recruited to growing daughter centrioles, and they strongly suggest that the Cnn and Spd-2 molecules that are recruited around mother centrioles during M-phase are not recruited there as part of a complex with Sas-4. We combined 3D-SIM with FRAP (Conduit et al, 2014b) and found that the Sas-4-GFP signal only recovered at a single foci (Figure 3A, t = 120 s to t = 280 s) We confirmed that this recovery occurred at the daughter centriole by performing a two-colour-3D-SIM-FRAP experiment in embryos co-expressing Sas-4-GFP and Asl-mCherry, as Asl forms a toroid around only the mother centrioles (Figure 3B, t = −30 s) (Novak et al, 2014). The previously described S-CAP complexes are either absent or present at very low levels in these embryo extracts
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