The centrosome is the major microtubule organizing center found in animals, being composed of two microtubule-based cylinders, the centrioles, which are surrounded by an electron-dense proteinaceous pericentriolar material (PCM) that nucleates microtubules. The centriole can also function as a basal body, nucleating cilia formation. Centrioles are normally formed in coordination with the cell cycle, one new centriole forming close to each existing one, within a PCM cloud (Bettencourt-Dias and Glover, 2007; Fu et al., 2015).

The first structure of the centriole to be assembled is the cartwheel, a ninefold symmetrical structure, composed of SAS-6, CEP135/Bld10, STIL/Ana2/SAS-5, amongst others (Kitagawa et al., 2011; Lin et al., 2013; Nakazawa et al., 2007; Tang et al., 2011; van Breugel et al., 2011). This is followed by centriole elongation through the deposition of centriolar microtubules which is dependent on components such as CPAP/SAS-4 (Kohlmaier et al., 2009; Schmidt et al., 2009; Tang et al., 2009). Remarkably, SAS-6 and CEP135/Bld10 can self-assemble in vitro to generate a ninefold symmetrical cartwheel (Guichard et al., 2017). In vivo, active PLK4 is known to recruit and phosphorylate STIL, which then recruits SAS-6 (Arquint et al., 2012; Arquint et al., 2015; Moyer et al., 2015; Ohta et al., 2014). In a process called ‘centriole-to-centrosome conversion’, the centriole recruits centriole-PCM linkers, such as CEP192/SPD2 and CEP152/asterless, which contribute to both centriole biogenesis and PCM recruitment (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010; Sonnen et al., 2013). Finally, CDK5RAP2/CNN and pericentrin are recruited, which then recruit and activate γ-tubulin and create an environment favorable for concentrating tubulin, leading to the formation of a proficient matrix for microtubule nucleation and organization (Delaval and Doxsey, 2010; Feng et al., 2017; Megraw et al., 2011; Woodruff et al., 2017).

The cascade of phosphorylation and interaction events between centriole components leading to centriole biogenesis is an intricate succession of positive feedback interactions. That circuit leads to amplification of an original signal present at the centriole, such as the presence of active PLK4, or its substrate STIL, hence perpetuating centriole biogenesis there (Arquint and Nigg, 2016; Lopes et al., 2015; Moyer et al., 2015). Additionally, the already existing ‘older’ centriole is surrounded by PCM, which could help to localize and concentrate centriole components during centriole assembly, reinforcing centriole formation close by. In support of this idea, when centrioles are eliminated in human cells by laser ablation, they form de novo within a PCM cloud (Khodjakov et al., 2002). Moreover, exaggeration of the PCM cloud by overexpressing a PCM component, pericentrin, in S-phase-arrested CHO cells, induces the formation of numerous daughter centrioles (Loncarek et al., 2008). Although pericentrin is not shown to be implicated in centriole biogenesis per se to date, dissociation of another pericentrin-related protein, AKAP450, from the centrosome, interferes with centriole duplication in human cells (Keryer et al., 2003). Moreover, PCM components, γ-tubulin and Spd-5, a functional analogue of Drosophila CNN and Hs Cdk5rap2, contribute to centriole assembly in C. elegans (Dammermann et al., 2004). Downregulation of the PCM in Drosophila, has been shown to lead to: i) fragmented and short centrioles, in the case of pericentrin (PLP) (Martinez-Campos et al., 2004; Roque et al., 2018), ii) disengaged centrioles, in the case of CNN (Lucas and Raff, 2007; Megraw et al., 2001), and iii) centriole disassembly, in the case of the removal of all PCM components (Pimenta-Marques et al., 2016). It is thus likely that the PCM plays critical roles in assembling and/or maintaining centriole structures. Despite the possible importance of the PCM, it is difficult to study its roles on centrioles due to confounding effects of the many centriole-centriole component interactions that exist in centriole-containing animal cells.

To define the contribution of PCM in regulating centriole components, we explored a system that has no endogenous centriole components, taking advantage of the diverged centrosomes observed in nature. While centrioles are ancestral in eukaryotes, they were lost in several branches of the eukaryotic tree, concomitantly with the loss of the flagellar structure (Carvalho-Santos et al., 2010). Instead of a canonical centrosome, yeasts have a spindle pole body (SPB), a layered structure composed of a centriole-less scaffold that similarly recruits γ-tubulin and other PCM components and nucleates microtubules (Cavanaugh and Jaspersen, 2017). The timing and regulation of SPB biogenesis are similar to the one observed in animal centrosomes (Lim et al., 2009; Kilmartin, 2014; Rüthnick and Schiebel, 2016). It is likely that the animal centrosome and yeast SPB evolved from a common ancestral structure that had centrioles (Figure 1A), as early-diverged basal fungi such as chytrids (e.g. Rhizophydium spherotheca) have a centriole-containing centrosome (Powell, 1980). By expressing animal centriole components in fission yeast, we observed that the SPB recruits them. We further demonstrate that the SPB conserved PCM component Pcp1/pericentrin recruits the centriole component SAS-6. We further validated this interaction in animals and show it is important for centriole elongation. Our work reveals an important role for pericentrin in recruiting centriole components and regulating centriole structure in animals.

Figure 1

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List of the predicted orthologues of the human centrosome and S. pombe SPB components in animal and fungi species.

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We first investigated the conservation of centrosome components, searching for orthologues of the known animal proteins comprising: components of centrioles that are required for centriole biogenesis (SAS-6, STIL/Ana2/SAS-5, CPAP/SAS-4, CEP135/Bld10 and CEP295/Ana1), linkers of the centriole to the PCM, which are bound to the centriole and are required for PCM recruitment (CEP152 and CEP192), and the PCM itself, which is involved in γ-tubulin recruitment and anchoring (pericentrin, Cdk5rap2 and γ-tubulin itself). In addition, to better understand when the SPB originated, we also searched for orthologs of the fission yeast SPB components: the core scaffold proteins (Ppc89, Sid4, Cdc11, Cut12 and Cam1; Bestul et al., 2017; Chang and Gould, 2000; Krapp et al., 2001; Moser et al., 1997; Rosenberg et al., 2006) and the half-bridge proteins (Sfi1 and Cdc31; Kilmartin, 2003; Paoletti et al., 2003), which are required for SPB duplication.

Consistent with previous studies (Carvalho-Santos et al., 2010; Hodges et al., 2010), the proteins required for centriole biogenesis in animals were not identified in the fungal genomes, with exception of chytrids, which have centrioles (Figure 1B). Centriole-PCM connectors were not found in both chytrids and yeasts. In contrast, when investigating the PCM composition, pericentrin and Cdk5rap2 were found in all fungal species, although the budding yeast Spc110 and Spc72 only share short conserved motifs with pericentrin and Cdk5rap2, respectively (Lin et al., 2014).

Regarding the SPB components, we found that Cam1 and Cdc31 are both highly spread across opisthokonts, which may reflect a conserved module or, alternatively an MTOC-independent conserved function of these proteins. On the other hand, we could only find proteins such as Ppc89 and Sid4 in yeasts, but not in chytrids, suggesting that some of the yeast-specific structural building blocks of the SPB appeared after branching into yeasts (Figure 1B).

Altogether, these results suggest the yeast centrosome is very different from the animal canonical centrosome, not having centriole and centriole-PCM adaptors, and being composed of several yeast-specific SPB components. Our results suggest that different proteins are involved in assembling different modules of the centrosome and that they can be lost when that module is lost, leading to a divergence of the remaining structures. Importantly, while centriole components were lost, the PCM module is conserved in animals and fungi in terms of composition and function, establishing fission yeast as an interesting system to study the interaction between components of the centriole and the PCM. We thus expressed key animal centriole components in fission yeast and asked whether they would interact with the PCM at the SPB or other yeast microtubule organizing centers.

Animal centriole components localize to the fission yeast SPB

We used individual Drosophila centriole components as they are well-characterized and tested their localization when expressed in fission yeast. We chose five critical components, SAS-6, CEP135/Bld10, CPAP/SAS-4, STIL/Ana2/SAS-5 and PLK4 for the test. All genes coding for these proteins are absent from the yeast genomes (Figure 1B). Fission yeast SPBs are easily recognizable under light microscopy with fluorescent protein-tagged SPB marker proteins, such as Sid4 and Sfi1 (Chang and Gould, 2000; Kilmartin, 2003), which show distinct localization as a clear focus. Therefore, we examined if the animal centriole proteins could recognize and thus localize to the SPB.

GFP or YFP-tagged Drosophila centriole proteins were heterologously expressed under control of the constitutive atb2 promoter (Matsuyama et al., 2008) or the inducible nmt1 promoter (Maundrell, 1990) in fission yeast. Despite the one billion years separating yeasts from animals (Douzery et al., 2004; Parfrey et al., 2011), SAS-6-GFP, Bld10-GFP and YFP-SAS-4 co-localized with fission yeast Sid4 to the SPB (Figure 2A and B). In addition to the localization on the SPB, YFP-SAS-4 signal was also weakly observed along interphase cytoplasmic microtubules, likely reflecting its microtubule-binding capacity (Gopalakrishnan et al., 2011). In contrast, GFP-Plk4 and YFP-Ana2 did not localize to the SPB and existed as foci in the cytoplasm (Figure 2A and B). We confirmed the expression of the fusion proteins with the expected sizes (Figure 2—figure supplement 1A). Cells expressing centriole proteins which localize to the SPB grew as well as control cells, which have no centriole protein (Figure 2—figure supplement 1B), suggesting their expression does not impair yeast growth.

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The surprising result that SAS-6, Bld10 and SAS-4 independently localize to the SPB, suggests that one or more fission yeast SPB component can recruit them. These results indicate that SPBs and canonical centrosomes have diverged less than what would be expected from their diverse morphology and divergent protein composition.

Fission yeast Pcp1 is required for SAS-6 recruitment

Next, we examined if Pcp1 is required for localization of SAS-6, Bld10 and SAS-4 on the SPBs using a temperature-sensitive mutant of Pcp1 (pcp1-14), in which the amount of Pcp1 protein is already reduced, and further reduced when grown at the restrictive temperature (Tang et al., 2014). These cells also arrest in mitosis when at the restrictive temperature. To compare the signal intensity in cells at the same cell cycle stage, mitosis, we introduced the cut7-446 allele both in wild-type and pcp1-14 background. Cut7 is a mitotic kinesin, its mutation fails in interdigitating the mitotic spindle and causes the cells to arrest in early mitosis (Hagan and Yanagida, 1990). Hereafter, we refer to the strains with cut7-446 and pcp1-14 cut7-446 alleles, as the control and pcp1 mutant, respectively. The intensity of SAS-6-GFP per SPB was significantly increased in control cells when arrested in prometaphase (Figure 3A and B). We observed reduced SAS-6-GFP intensity in the pcp1 mutant both at the permissive and restrictive temperatures (Figure 3A and B). This indicates that Pcp1 is required for SAS-6 recruitment to the SPB. Unlike SAS-6, Bld10-GFP intensity was not reduced, but slightly increased in the pcp1 mutant (Figure 3C and D). It is possible that Bld10 is recruited to the SPB by another SPB component(s) which is upregulated in the pcp1 mutant. Although the intensity of SAS-4 was reduced in the pcp1 mutant (Figure 3E and F), we found that the total protein of YFP-SAS-4 was also lower in the pcp1 mutant while that of SAS-6-GFP and Bld10-GFP was comparable in control and pcp1 mutant lysates (Figure 3G). We think that YFP-SAS-4 might be stabilised by Pcp1 in fission yeast cells, and therefore we cannot conclude whether Pcp1 is required for YFP-SAS-4 localization on the SPB. Since SAS-6 is such a critical component in centriole assembly, and its localization is determined by Pcp1, we decided to explore further how SAS-6 is recruited to the SPB by Pcp1.

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The source data to plot the graphs in Figure 3B,D,F.

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It is known in Drosophila and human cells that phosphorylation and interaction of PLK4 with STIL/Ana2, facilitates STIL recruitment to the centriole and its interaction and recruitment of SAS-6 (Arquint et al., 2012; Moyer et al., 2015; Ohta et al., 2014; Vulprecht et al., 2012). However, neither Plk4 nor STIL are present in the fission yeast genome (Figure 1B), suggesting that Pcp1/pericentrin is part of an additional and ancestral molecular pathway for SAS-6 recruitment.

SAS-6 interacts with the conserved region of Pcp1, and Pcp1 is sufficient to recruit SAS-6

We reasoned that if the interaction between SAS-6 and Pcp1/pericentrin is an ancient and conserved connection, they should interact through an evolutionarily conserved domain in Pcp1. Subsequently, we determined which part of Pcp1 is required for its interaction with SAS-6. Full-length and truncation mutants of Pcp1 (N, M and C) were co-expressed with SAS-6-GFP (Figure 4A). Only full-length Pcp1 and its C-terminal region containing the conserved PACT domain interacted strongly with SAS-6 (Figure 4B). Given that the Pcp1-M fragment was weakly expressed, we cannot exclude completely the possibility that it also interacts with SAS-6-GFP (Figure 4B). The PACT domain localizes to the centriole wall in animals and is required for MTOC targeting both in animal and fungi (Gillingham and Munro, 2000).

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The source data to plot the graph in Figure 4E.

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We next asked whether Pcp1 could recruit SAS-6 ectopically. It is known that Pcp1 overexpression forms multiple Pcp1-containing foci in the cytoplasm (Flory et al., 2002). We thus examined if the cytoplasmic Pcp1 foci recruit SAS-6 (illustrated in Figure 4C). Overexpressed mCherry-tagged Pcp1 recruited SAS-6-GFP, but not GFP (control), to such foci (Figure 4D and E), indicating that Pcp1 can ectopically recruit SAS-6.

Though epifluorescence micrographs suggest SAS-6 co-localizes with Sid4 and Pcp1 (Figures 2 and 4), due to the resolution limit of a conventional optical microscope (~200 nm), we failed to conclude precisely where SAS-6 localizes to. To further determine the precise localization of SAS-6, we analyzed the relative position of SAS-6-GFP with respect to core SPB components Pcp1-tdTomato or Sid4-tdTomato (Figure 4—figure supplement 1A) by structured illumination microscopy (SIM). The foci of Pcp1-tdTomato and Sid4-tdTomato within the duplicated SPBs were distinguishably separated (60 ± 10 nm) (Figure 4—figure supplement 1B and C). Importantly, SAS-6-GFP center of mass localizes at ~50 nm distance from both Pcp1-tdTomato and Sid4-tdTomato center of mass. Given that the diameter and height of SPB is 180 nm and 90 nm respectively (Ding et al., 1997), our data suggest that ectopically expressed SAS-6-GFP localizes to the core of the SPB.

Conservation of the Pcp1/pericentrin - SAS-6 interaction

Our experiments suggest that there are conserved interactions between centrioles and pericentrin, whose evolution is constrained. They further suggest that pericentrin has an important role in recruiting centriole components. To test our prediction, we examined in animal cells whether pericentrin interacts with SAS-6 and helps to recruit it to the centriole.

The pericentrin family varies in protein length but contains the conserved PACT domain in the C-terminal region (Figure 5A). Since we observed that fission yeast Pcp1 interacts with SAS-6 through the PACT-domain containing region, we tested whether SAS-6 interacts with the PACT domain of Drosophila pericentrin ortholog, pericentrin-like protein (PLP). EGFP-tagged SAS-6 and HA-tagged PLP fragment containing the conserved PACT domain were co-expressed in Drosophila melanogaster tissue cultured cells (D.Mel cells). Consistent with the results obtained in fission yeast, SAS-6 interacts with the PACT domain (Figure 5B). To verify whether this interaction is direct, we validated this result in vitro, by performing in vitro binding assays using purified GST, GST-tagged SAS-6 and His-tagged PACT. GST-SAS-6 was specifically bound to His-PACT, indicating a direct interaction between these two proteins (Figure 5C).

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The source data to plot the graphs in Figure 5E and G.

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SAS-6 is recruited to the centriole by two complementary pathways

In animal cells, it is known that STIL/Ana2/SAS-5 recruits SAS-6 to the centriole (Arquint et al., 2012; Ohta et al., 2014; Moyer et al., 2015). However, it has been observed that STIL depletion does not completely prevent HsSAS-6 recruitment, and contribution of STIL does not fully account for centrosomal targeting of HsSAS-6 during interphase (Arquint et al., 2012; Keller et al., 2014). These results suggest that other factor(s) may also recruit SAS-6 in animals. Since overexpression of PACT recruits more SAS-6, we examined if PLP has a role in recruiting SAS-6 to the centriole in addition to STIL/Ana2. We performed a single depletion of PLP and Ana2 and a co-depletion of both (Ana2 and PLP) in D.Mel cells. We used mCherry depletion as a negative control for this experiment. Firstly, we investigated if SAS-6 recruitment is impaired by Ana2 and PLP RNAi. We focused on mitotic cells to compare all cells at the same cell cycle stage. The intensity of SAS-6 per centrosome was quantified in each RNAi condition. Upon depletion of Ana2 or PLP, SAS-6 intensity per pole was significantly reduced compared to control (Figure 6A and B). Moreover, double depletion of Ana2 and PLP caused an additive reduction of SAS-6 intensity. This result suggests SAS-6 is recruited by two complementary pathways, which depend on Ana2 and PLP. Western blotting analysis confirmed that the targeted proteins were efficiently depleted, while total SAS-6 protein levels were comparable in all conditions (Figure 6C). Considering that depletion of SAS-6 leads to a reduction in centriole number in both D.Mel cells and the fly (Dobbelaere et al., 2008; Rodrigues-Martins et al., 2007), we asked whether altered recruitment of SAS-6 by depleting Ana2 and PLP could also affect centriole biogenesis. We observed that co-depletion of Ana2 and PLP leads to stronger reduction in the number of centrioles as compared to the single depletions, indicating both pathways contribute to centriole biogenesis (Figure 6D,E, and Figure 6—figure supplement 1).

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The source data to plot the graph in Figure 6B and E.

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Furthermore, we asked if the centriole number reduction observed in PLP-depleted cells is attributable to PLP function or is an off-target effect, through a rescue experiment. We first depleted the endogenous PLP using another dsRNA against the 3’UTR region (3’UTR PLP RNAi) and expressed EGFP-tagged full-length PLP. Similar to Figure 4D and E, 3’UTR PLP RNAi efficiently depleted endogenous PLP protein and caused a significant reduction in centriole number (Figure 6—figure supplement 2). Since we treated the cells with 3’UTR PLP RNAi for two rounds aiming at a more efficient protein removal, the percentage of cells with reduced centriole number was slightly higher than in former PLP RNAi used for Figure 6. In contrast, when we expressed full-length PLP after PLP depletion, the centriole number defect was rescued, indicating the specific contribution of PLP for centriole formation (Figure 6—figure supplement 2). Moreover, we noticed that expression of full-length PLP in the presence of endogenous PLP induced centriole amplification similar to PACT overexpression (Figure 6—figure supplement 2).

Drosophila pericentrin is required for SAS-6 recruitment and is important for centriole/basal body (BB) elongation in vivo

It is noteworthy that D.Mel cells are a sensitized system in which depletion of centriole components often shows an enhanced phenotype as compared to what is observed in the whole organism (the fly). For example, depletion of Bld10/CEP135 and CP110 by RNAi in D.Mel cells leads to a reduction in centriole number and length, respectively (Carvalho-Santos et al., 2010; Delgehyr et al., 2012), but the Drosophila mutants of these proteins only exhibit mild centriole defects (Carvalho-Santos et al., 2010; Franz et al., 2013; Roque et al., 2012). In previous studies using PLP-mutant flies, centriole number was not reduced, but centrioles were recently observed to be shorter in wing disc cells (Martinez-Campos et al., 2004; Roque et al., 2018), suggesting an underappreciated generic role of PLP in centriole structure.

To further investigate the role of pericentrin in SAS-6 recruitment and centriole biogenesis we focused on fly spermatogenesis, as centrioles are converted to basal bodies to form cilia and elongate to more than 1 μm, becoming highly visible and easy to study. To investigate this, we depleted PLP during centriole assembly and basal body maturation in spermatocytes (PLPRNAi) using Gal4^Bam (Chen and McKearin, 2003; see the timeline in Figure 7A). We studied its consequences in the localization of basal body components and basal body structure and, subsequently, in male fertility (Figure 7). The knockdown of PLP by RNAi did not affect centriole number in sperm cells (Figure 7B and C), suggesting that the STIL/Ana2 pathway recruits sufficient SAS-6 to initiate centriole biogenesis. However, PLP RNAi led to a decrease in centriole length, as observed with the PLP mutant in somatic cells (Roque et al., 2018), and to male infertility. Our laboratory has recently shown that SAS-6 is important for centriole elongation (Jana et al., 2018). We wondered whether PLP is necessary for recruiting a pool of SAS-6 that is needed for centriole elongation. Remarkably, PLP RNAi affected SAS-6 recruitment to the BBs (Figure 7D and E). Altogether, these results indicate that PLP is involved in sperm BB elongation by recruiting SAS-6, a phenotype that is further accentuated in tissue culture cells.

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The source data to plot the graph in Figure 7C and E.

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The Calmodulin-PACT conserved interaction is likely to constrain the evolution of the PACT domain

We showed that the PACT domain has the capacity to interact with SAS-6 both in fission yeast and Drosophila cells, and the interaction between SAS-6 and pericentrin through PACT contributes to centriole assembly and elongation. It is possible that this interaction is ancestral, linking the two modules, the centriole and the PCM before the split between animals and fungi occurred one billion years ago. However, given the lack of centrioles and the divergence of the PCM in yeasts, we wondered why the PACT domain has retained the SAS-6 interaction surface. We hypothesized that other protein(s) that interact with the same region of the PACT domain as SAS-6, could be constraining the evolution of the interacting surface.

The PACT domain contains two highly conserved calmodulin (CaM)-binding domains (CBD1 and CBD2) (Galletta et al., 2014, Figure 8A). The interaction of pericentrin with calmodulin at the CaM-binding domain is conserved and is important for its function both in animals and yeasts. In Drosophila, this interaction controls the targeting of PLP to the centrosome and is, therefore, critical for its function (Galletta et al., 2014). The interaction of the budding yeast pericentrin, Spc110, with calmodulin, is required for the SPB to nucleate microtubules and to form the spindle (Kilmartin and Goh, 1996; Spang et al., 1996; Stirling et al., 1994; Stirling et al., 1996). Therefore, we asked whether SAS-6 interacts with a conserved PLP segment, starting just before CBD1 and ending just after CBD2, hereafter called CBD (see Figure 8A in yellow). Similarly, as in Figure 5B, we co-expressed EGFP-tagged SAS-6 and the HA-tagged CBD in D.Mel cells to examine their interaction. Indeed, similar to the PACT domain, CBD interacted with SAS-6-EGFP (Figure 8B). This result indicates that this conserved segment within the PACT domain is sufficient for interaction with SAS-6.

Figure 8

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Next, we asked whether SAS-6 is present in the same complex with calmodulin. Intriguingly, we found that SAS-6-EGFP, CBD and calmodulin formed a complex (Figure 8B). Since SAS-6-EGFP and calmodulin did not interact directly, we concluded that the complex forms through the CBD segment. Moreover, we observed that the CBD polypeptide was stabilised in the presence of SAS-6-EGFP, and this was more pronounced with the addition of calmodulin, suggesting that formation of this complex might stabilize pericentrin (Figure 8B). Since the binding of SAS-6 and calmodulin to the CBD was not competitive, it is likely that these two proteins interact with adjacent but distinct amino acids within the CBD. Further biochemical analysis will be necessary to estimate the exact stoichiometry of the protein complex.

Our result suggests that the fact that pericentrin function and stability has always relied on its interaction with calmodulin has constrained the evolution of the pericentrin´s CBD’s local structure, therefore retaining its affinity for SAS-6.

In this study, we examined the relationship between the PCM and centriole components, taking advantage of a heterologous system, the fission yeast, which does not have centrioles, and has lost the coding sequences for its components. Surprisingly, the Drosophila core centriole components SAS-6, Bld10 and SAS-4 localized to the fission yeast SPB. In particular, SAS-6 was specifically recruited to the SPB through its interaction with the conserved PCM component, Pcp1/pericentrin, via its PACT domain. Importantly, this interaction was also observed in animals (Drosophila). Further analysis revealed that pericentrin is required for both SAS-6 recruitment to centrioles in addition to the STIL/Ana2 pathway, and for proper centriole assembly, in particular, centriole elongation. It is estimated that animals and fungi diverged from their common ancestor about one billion years ago (Douzery et al., 2004; Parfrey et al., 2011). Our results reveal an evolutionarily conserved relationship between the centriole and the PCM, in which the PCM is needed for centriole component recruitment (Figure 8C). Therefore, the localization of new centrioles is likely to be dictated not just by positive regulatory feedbacks amongst centriole components, but also through the regulation of centriole component localization by the PCM.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files are provided for Figure 1, Figures 2, Figure 2-figure supplement 1, Figure 3, Figure 4, Figure 4-figure supplement 1-3, Figure 5, Figure 6, Figure 6-figure supplement 1-2, and Figure 7.

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Author details

1. Daisuke Ito

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing, Created fission yeast strains, Performed fission yeast experiments, Performed bioinformatics analyses, Performed cell biology in D.Mel cells and biochemistry experiments, Performed super-resolution imaging, Conceived the study, Analyzed data

   For correspondence

   itodaisuke1982@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6759-0901

2. Sihem Zitouni

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Data curation, Formal analysis, Investigation, Performed cell biology in D.Mel cells and biochemistry experiments

   Contributed equally with

   Swadhin Chandra Jana

   Competing interests

   No competing interests declared

3. Swadhin Chandra Jana

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Data curation, Formal analysis, Investigation, Performed super-resolution imaging, Performed Drosophila experiments

   Contributed equally with

   Sihem Zitouni

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8311-3849

4. Paulo Duarte

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Data curation, Formal analysis, Investigation, Created fission yeast strains

   Competing interests

   No competing interests declared

5. Jaroslaw Surkont

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Data curation, Formal analysis, Investigation, Performed bioinformatics analyses

   Competing interests

   No competing interests declared

6. Zita Carvalho-Santos

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Present address

   Champalimaud Centre for the Unknown, Lisbon, Portugal

   Contribution

   Data curation, Formal analysis, Investigation, Created fission yeast strains, Contributed to the conception

   Competing interests

   No competing interests declared

7. José B Pereira-Leal

   1. Instituto Gulbenkian de Ciência, Oeiras, Portugal
   2. Ophiomics, Precision Medicine, Lisboa, Portugal

   Contribution

   Supervision, Contributed to the conception

   Competing interests

   No competing interests declared

8. Miguel Godinho Ferreira

   1. Instituto Gulbenkian de Ciência, Oeiras, Portugal
   2. Institute for Research on Cancer and Aging of Nice (IRCAN), INSERM U1081 UMR7284 CNRS, Nice, France

   Contribution

   Supervision, Contributed to the conception

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8363-7183

9. Mónica Bettencourt-Dias

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing, Conceived the study, Analyzed data

   For correspondence

   mdias@igc.gulbenkian.pt

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1987-5598

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