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S41467-020-14767-2.Pdf ARTICLE https://doi.org/10.1038/s41467-020-14767-2 OPEN CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion ✉ Enrico S. Atorino 1, Shoji Hata 1, Charlotta Funaya2, Annett Neuner1 & Elmar Schiebel 1 Centrosomes are essential organelles with functions in microtubule organization that dupli- cate once per cell cycle. The first step of centrosome duplication is the daughter centriole 1234567890():,; formation followed by the pericentriolar material recruitment to this centriole. This maturation step was termed centriole-to-centrosome conversion. It was proposed that CEP295-dependent recruitment of pericentriolar proteins drives centriole conversion. Here we show, based on the analysis of proteins that promote centriole biogenesis, that the developing centriole structure helps drive centriole conversion. Depletion of the luminal centriole protein CEP44 that binds to the A-microtubules and interacts with POC1B affecting centriole structure and centriole conversion, despite CEP295 binding to centrioles. Impair- ment of POC1B, TUBE1 or TUBD1, which disturbs integrity of centriole microtubules, also prevents centriole-to-centrosome conversion. We propose that the CEP295, CEP44, POC1B, TUBE1 and TUBD1 centriole biogenesis pathway that functions in the centriole lumen and on the cytoplasmic side is essential for the centriole-to-centrosome conversion. 1 Zentrum für Molekulare Biologie, Universität Heidelberg, DKFZ-ZMBH Allianz, 69120 Heidelberg, Germany. 2 Electron Microscopy Core Facility, Universität ✉ Heidelberg, 69120 Heidelberg, Germany. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:903 | https://doi.org/10.1038/s41467-020-14767-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14767-2 he centrosome is the main microtubule-organising centre quantified the number of centrosomes counting the γ-tubulin foci T(MTOC) of higher eukaryotic cells1,2. This organelle (Fig. 1c). Only G1 cells (negative for EdU and Cenp-F24) comprises a central cylindrical tubulin-based structure, the (Supplementary Fig. 1b for Cenp-F) were considered in this centriole3, and a protein matrix that surrounds the centriole quantification in order to simplify the analysis. In the 72 h CEP44 called the pericentriolar matrix (PCM)4–6. The PCM is a highly knockdown sample, 80.4 ± 5.0% of G1 cells displayed less than organised toroid matrix in which the proteins that constitute it two defined γ-tubulin signals (Fig. 1b, c). Analysis of the presence are concentrically placed in it and emanate from the proximal of centrioles in CEP44 depleted G1 cells judging the number of end region of the centriole5. One of the main functions of the centrin1 foci, showed the loss of centrioles in 35.0 ± 2.8% of cells PCM is the microtubule (MT) nucleation7–9. The centriole is a (Supplementary Fig. 1c, d). cylindrical structure made up of MT-triplets arranged in a 9-fold The clear stronger impact on γ-tubulin than on radial symmetry10,11. Each triplet consists of an inner tubule of 13 centrin1 suggested that the PCM recruitment defect arises before tubulin protofilaments (the A tubule) to which two additional the centriole loss. We explored this possibility further at an earlier tubules, the B and then the C tubules, are attached12. time point of CEP44 depletion (60 h) when the centriole loss in Centrosomes duplicate once per cell cycle. The newly born G1 was minimal (5.1 ± 1.6, Supplementary Fig. 1e, f). 60% of the daughter centriole (dC) grows perpendicularly on the mother cells still showed a strong defect in the recruitment of both PCM centriole (mC) and is kept engaged on it until the cell enters the components γ-tubulin and PCNT, which corresponded in its next cell cycle13. During the process of the dC elongation, its magnitude with the CEP44 loss (Fig. 1d–f; Supplementary structure grows radially and builds MT triplets via TUBE1 and Fig. 1e–i). Moreover, the PCM recruitment defect associated TUBD114. with the centrioles lacking the mC marker CEP16425, thus with After centriole formation in S/G2 phase, the dC is converted the dCs (Supplementary Fig. 1j–l). into a centrosome by the step-wise recruitment of PCM pro- We explored whether the loss of the centrioles in G1 after 72 h teins. This process called centriole-to-centrosome conversion depletion (Fig. 1b) was due to a centriole duplication defect or (CCC)15,16 is essential for the new centrosome to gain MT instability of the centrioles. Therefore we tested the duplication of nucleation activity and the ability to duplicate. The current centriole in G2 cells at 60 h upon CEP44 depletion by counting model, derived from D. melanogaster and different human the number of the foci of the centriolar marker CEP9726 (4 or cultured cell lines, suggests a PCM protein recruitment cascade fewer). We observed that 30.3 ± 4.1% of G2 cells in the on the dC wall. The centriole proximal end protein CEP135 siCEP44 sample showed <4 CEP97 foci, thus a defect in binds to the centriole wall and connects to CEP295, a key the ability of the centrioles to duplicate (Fig. 1g and h), explaining proteininCCC.CEP295recruits the PLK4 kinase adaptor the loss of centrioles in the subsequent G1 phase at 72 h CEP152 and the γ-tubulin recruitment factor CEP192 to the (Supplementary Fig. 1c and d). In all following experiments we new dC16,17. In human cells CCC was described to stabilise the depleted CEP44 for 60 h and analysed G1 cells in order to avoid a centrioles in mitosis15. Therefore, loss of CCC impairs centro- centriole duplication defect. This together indicates that CEP44 some homeostasis18 and causes developmental defects, e.g. primarily plays a role in the CCC of the dC and not in the microcephaly, ciliopathies and cancer19–21. maintenance of mother centrosomes. How CCC works on a molecular level is little understood. We confirmed that CEP44 was responsible for the CCC Interestingly, the processes of centriole maturation and CCC phenotype. We rescued the defect of dCs to recruit PCM by occur simultaneously raising the possibility of interplay between expressing an exogenous siRNA-resistant CEP44 version, which the unique 9-fold MT-triplet structure and PCM recruitment to completely restored centrosome function upon siCEP44 treat- the dC. To better understand the CCC mechanisms we focus on ment of the cells (Supplementary Fig. 1m, n). In addition, we centrosome biogenesis studying CEP44, an essential centrosomal analysed CEP44 knockout (KO) cells that were constructed by a protein22,23. Our analysis identifies CEP44 as a luminal centriolar CRISPR/Cas9 approach27. Bi-allelic CEP44 KOs were only protein that binds to A-MTs and the inner centriole protein obtained in a RPE1 p53−/− background cell line (Supplementary POC1B. CEP44 depletion impairs CCC although the binding of Fig. 2a, b), which is consistent with a function of CEP44 in CEP295 to the dC is unaffected indicating additional CCC CCC23. The KO cell lines showed the same PCM recruitment mechanisms than the CEP295-dependent recruitment of PCM phenotype as described for the CEP44 siRNA-induced knock- proteins. Interestingly, also depletion of POC1B, TUBE1 and down (Supplementary Fig. 2c–e). Similarly to the knockdown TUBD1 affects CCC beside centriole structure. We therefore rescue data, we were able to restore proper converted centrosome propose that centriole biogenesis via CEP295, CEP44, POC1B, number by expressing exogenously CEP44 in CEP44−/− p53−/− TUBE1 and TUBD1 pathway is important for the conversion of cells (Supplementary Fig. 2c–e). centrioles to fully-functional centrosomes. CEP44 depletion leads to MT nucleation and mitotic defects.A Results defect in γ-tubulin recruitment should affect the MTOC activity CEP44 is a centriole protein necessary for the CCC mechanism. of centrosomes. RPE1 C-Nap1 (KO) cells28 were exploited for this Our interest in understanding the biogenesis and the function of defect because their non-cohered centrosomes allowed us to the centrosome drove us to characterise CEP44 out of novel discern the MTs generated from the mother and the daughter centrosomal proteins22 because it was the only of these that was centrosomes and thus to quantify them. CEP44-depleted described to be essential in human cells23.Affinity purified daughter centrosomes displayed reduced MT nucleation activity antibodies rose against the protein showed that in G1 and S phase in a MT regrowth assay compared to control daughter centro- (EdU positive) cells, CEP44 localised to mCs whereas in G2 cells somes (Fig. 1i, j) consistent with the failure of dCs to recruit γ- it appeared as 4 foci localising to all 4 centrin1 signals, the two tubulin and PCNT (Fig. 1f and Supplementary Fig. 1h). As a mCs and two dCs (Fig. 1a). This led us to conclude that CEP44 is consequence, CEP44 depletion severely compromised the ability a centriolar protein. of cells to assemble symmetrically balanced mitotic spindle poles In order to understand the function of CEP44 at centrioles, we (Fig. 1k, l). We conclude that deficiency in CEP44 recruitment to knocked down the expression of CEP44 in RPE1 h-TERT cells via centrosomes perturbs the homeostasis of centrioles and the siRNA for 72 h (Fig. 1b and Supplementary Fig. 1a–d) and organisation of mitotic spindle poles. 2 NATURE COMMUNICATIONS | (2020) 11:903 | https://doi.org/10.1038/s41467-020-14767-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14767-2 ARTICLE a Centrin1 CEP44
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