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Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9 fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.

Formation of a single new centriole from a pre-existing centriole is strictly controlled to maintain correct centrosome number and spindle polarity in cells. However, the mechanisms that govern this process are incompletely understood. Here, using several human cell lines, immunofluorescence and structured illumination microscopy methods, and ubiquitination assays, we show that the E3 ubiquitin ligase F-box and WD repeat domain–containing 7 (FBXW7), a subunit of the SCF ubiquitin ligase, down-regulates spindle assembly 6 homolog (HsSAS-6), a key protein required for procentriole cartwheel assembly, and thereby regulates centriole duplication. We found that FBXW7 abrogation stabilizes HsSAS-6 and increases its recruitment to the mother centriole at multiple sites, leading to supernumerary centrioles. Ultrastructural analyses revealed that FBXW7 is broadly localized on the mother centriole and that its presence is reduced at the site where the HsSAS-6–containing procentriole is formed. This observation suggested that FBXW7 restricts procentriole assembly to a specific site to generate a single new centriole. In contrast, during HsSAS-6 overexpression, FBXW7 strongly associated with HsSAS-6 at the centriole. We also found that SCF(FBXW7) interacts with HsSAS-6 and targets it for ubiquitin-mediated degradation. Further, we identified putative phosphodegron sites in HsSAS-6, whose substitutions rendered it insensitive to FBXW7-mediated degradation and control of centriole number. In summary, SCF(FBXW7) targets HsSAS-6 for degradation and thereby controls centriole biogenesis by restraining HsSAS-6 recruitment to the mother centriole, a molecular mechanism that controls supernumerary centrioles/centrosomes and the maintenance of bipolar spindles.

Over the course of evolution, the function of the centrosome has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, as illustrated by the presence of centrioles in humans and a spindle pole body in yeast (SPB). Consistently, the composition of these two core elements has diverged greatly, with the exception of centrin, a protein known to form a complex with Sfi1 in yeast to structurally initiate SPB duplication. Even though SFI1 has been localized to human centrosomes, whether this complex exists at centrioles and whether its function has been conserved is still unclear. Here, using conventional fluorescence and super-resolution microscopies, we demonstrate that human SFI1 is a bona fide centriolar protein localizing to the very distal end of the centriole, where it associates with a pool of distal centrin. We also found that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the specific loss of the distal pool of centrin, without altering centriole duplication in human cells, in contrast to its function for SPB. Instead, we found that SFI1/centrin complexes are essential for correct centriolar architecture as well as for ciliogenesis. We propose that SFI1/centrin complexes may guide centriole growth to ensure centriole integrity and function as a basal body.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers / distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the apparent evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL and OFD1 form an evolutionary conserved complex that is crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early after centriole duplication on the external surface of the procentriole and define the future location of the distal appendages. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of this complex for distal appendage assembly and basal body docking. Finally, we show that mammals require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruits the distal appendage protein CEP83. Altogether, we propose that this ternary complex is required to determine the future position of distal appendages.

Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique kinase that utilizes its autophosphorylated noncatalytic cryptic polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4’s dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer., Regulation of centriole duplication is essential for normal chromosome segregation and the maintenance of genomic stability, and Polo-like kinase 4 (Plk4) is a known regulator with unclear mechanisms. Here the authors show that Plk4 undergoes phase separation into an assembling body through autophosphorylation which drives centriole biogenesis.

International audience; Reproductive and respiratory organs, along with brain ventricles, are lined by multiciliated epithelial cells (MCC) that generate cilia-powered fluid flows. MCC hijack the centrosome duplication pathway to form hundreds of centrioles and nucleate motile cilia. In these cells, the large majority of procentrioles are formed associated with partially characterized organelles called deuterosomes. We recently challenged the paradigm that deuterosomes and procentrioles are formed de novo by providing data, in brain MCC, suggesting that they are nucleated from the pre-existing centrosomal younger centriole. However, the origin of deuterosomes and procentrioles is still under debate. Here, we further question centrosome importance for deuterosome and centriole amplification. First, we provide additional data confirming that centriole amplification occurs sequentially from the centrosomal region, and that the first procentriole-loaded deuterosomes are associated with the daughter centriole or in the centrosomal centriole vicinity. Then, to further test the requirement of the centrosome in deuterosome and centriole formation, we depleted centrosomal centrioles using a Plk4 inhibitor. We reveal unexpected limited consequences in deuterosome/centriole number in absence of centrosomal centrioles. Notably, in absence of the daughter centriole only, deuterosomes are not seen associated with the mother centriole. In absence of both centrosomal centrioles, procentrioles are still amplified sequentially and with no apparent structural defects. They seem to arise from a focal region, characterized by microtubule convergence and pericentriolar material (PCM) assembly. The relevance of deuterosome association with the daughter centriole as well as the role of the PCM in the focal and sequential genesis of centrioles in absence of centrosomal centrioles are discussed.

Control of centrosome assembly is critical for cell division, intracellular trafficking and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule cytoskeleton, centrosome and centriolar satellites. SON is required for the proper splicing and expression ofCEP131which encodes a major centriolar satellite protein and is required to organize the trafficking and microtubule network around the centrosomes. This study highlights the importance of the distinct microtubule trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development.

Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a centriole to a dot-like morphology at the procentriole assembly site and this event is central for inducing centriole biogenesis. However, the detailed mechanisms underlying Plk4's capacity to drive its symmetry-breaking ring-to-dot relocalization remain largely unknown. Here, we showed that Plk4 self-initiates this process in an autophosphorylation-dependent manner and that STIL, its downstream target, is not required for this event. Time-dependent analyses with mEOS-fused photoconvertible Plk4 revealed that a portion of ring-state Plk4 acquires a capacity, presumably through autophosphorylation, to linger around a centriole, ultimately generating a dot-state morphology. Interestingly, Plk4 WT, but not its catalytically inactive mutant, showed the ability to form a nanoscale spherical assembly in the cytosol of human cells or heterologous E. coli, demonstrating its autophosphorylation-dependent self-organizing capacity. At the biochemical level, Plk4 - unlike its N-terminal βTrCP degron motif - robustly autophosphorylated the PC3 SSTT motif within its C-terminal cryptic polo-box, an event critical for inducing its physical clustering. Additional in vivo experiments showed that although STIL was not required for Plk4's initial ring-to-dot conversion, coexpressed STIL greatly enhanced Plk4's ability to generate a spherical condensate and recruit Sas6, a major component of the centriolar cartwheel structure. We propose that Plk4's autophosphorylation-induced clustering is sufficient to induce its ring-to-dot localization conversion and that subsequently recruited STIL potentiates this process to generate a procentriole assembly body critical for Plk4-dependent centriole biogenesis.

The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled–coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo . Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2–Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.

Panda et al. find that Rcd4 is required for centriole duplication and ciliogenesis in neurosensory organs but dispensable for spermatogenesis, indicating tissue specific requirements for centriole formation. Loading of Rcd4 and its binding partner Ana3 onto daughter centrioles is a prerequisite for the mitotic conversion of centrioles into centrosomes., Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. Here, we show that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. We find that neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.

Centriole duplication occurs once per cell cycle. Since only a single daughter centriole is assembled adjacent to each mother centriole, symmetry around the mother centriole must be broken in the process of centriole duplication. Recent studies have established that Plk4, a master kinase for centriole duplication, can self-assemble into condensates, and have suggested that this Plk4 self-assembly is the key to symmetry breaking. Here, we present the current hypotheses for how Plk4 could break symmetry around the mother centriole via autonomous regulation. After this initial symmetry-breaking process, the ring-to-dot conversion of Plk4 around the mother centriole completes the selection of the site for procentriole formation. We also discuss how this dynamic transition contributes to the strict regulation of centriole duplication.

Centrioles are polarized microtubule‐based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS‐6 proteins. A cryo‐tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub‐tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A‐C linker. Moreover, we report ~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS‐6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS‐6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms., Ultrastructural studies of centrioles from termite flagellates delineate the architecture of the centriole proximal region, including new insights into stacking of cartwheel‐forming SAS‐6 rings.

Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar cylindrical architecture, and this event is critical for the orderly recruitment of Plk4, a key regulator of centriole duplication. However, the way in which Cep57 interacts with the Cep63-Cep152 complex and contributes to the structure and function of Cep63-Cep152 self-assembly remains unknown. We demonstrate that Cep57 interacts with Cep63 through N-terminal motifs and associates with Cep152 via Cep63. Three-dimensional structured illumination microscopy (3D-SIM) analyses suggested that the Cep57-Cep63-Cep152 complex is concentrically arranged around a centriole in a Cep57-in and Cep152-out manner. Cep57 mutant cells defective in Cep63 binding exhibited improper Cep63 and Cep152 localization and impaired Sas6 recruitment for procentriole assembly, proving the significance of the Cep57-Cep63 interaction. Intriguingly, Cep63 fused to a microtubule (MT)-binding domain of Cep57 functioned in concert with Cep152 to assemble around stabilized MTs in vitro. Thus, Cep57 plays a key role in architecting the Cep63-Cep152 assembly around centriolar MTs and promoting centriole biogenesis. This study may offer a platform to investigate how the organization and function of the pericentriolar architecture are altered by disease-associated mutations found in the Cep57-Cep63-Cep152 complex.

Centriole reduplication leads to the formation of supernumerary centrosomes, which promote cellular transformation, invasion and are a hallmark of tumors. A close association between a mother centriole and a procentriole (engagement), established during centriole duplication, intrinsically blocks reduplication. Premature loss of centriole association predisposes centrioles for reduplication and occurs during various types of cell cycle arrests in the presence of high Polo-like kinase 1 activity. Here we use nano-scale imaging and biochemistry to reveal the processes leading to the loss of centriole association and reduplication. We discover that centriole reduplication is driven by events occurring on procentriole microtubule walls. These events are mechanistically different from mitotic centriole separation driven by Pericentrin and Separase but are similar to the physiological process of centriole distancing occurring in unperturbed cycling G2 cells. We propose a concept in which centriole reduplication is a consequence of hijacked and amplified centriole maturation process.HighlightsSeparase-mediated Pericentrin reorganization is not required for centriole distancing and reduplication in interphase.Expression of active Plk1 in S phase leads to centrosomal ultrastructural changes resembling G2 phase.Procentrioles without microtubule walls cannot disengage.Centriole distancing is intrinsically regulated by the events occurring on procentriole microtubules.

Cep152 and Cep63 act as the cradle and recruit Plk4 to initiate the centriole biogenesis in a mother centriole dependent manner. However, how the Cep152-Cep63 complex is targeted to the proximal end of mother centrioles is unclear. In this study, we show that Cep57 and its paralog, Cep57l1, colocalize with Cep63 and Cep152 at the proximal end of mother centrioles in both cycling cells and differentiated multiciliated cells. Both Cep57 and Cep57l1 associate with the Cep152-Cep63 complex by directly binding to the centrosomal targeting region of Cep63. Co-depletion of Cep57 and Cep57l1 blocks the loading of Cep63-Cep152 to the centriole and subsequently prevents centriole duplication. We propose that Cep57 and Cep57l1 act together to ensure the recruitment of the Cep63-Cep152 complex to the mother centrioles for procentriole formation.Summary statementCep57 and its paralog, Cep57l1, cooperatively act as a molecular scaffold to recruit the Cep63-Cep152 cradle to the proximal end of centrioles in the early stages of centriole duplication.

The centriole is a beautiful microtubule-based organelle that is critical for the proper execution of many fundamental cellular processes, including polarity, motility, and division. Centriole biogenesis, the making of this miniature architectural wonder, has emerged as an exemplary model to dissect the mechanisms governing the assembly of a eukaryotic organelle. Centriole biogenesis relies on a set of core proteins whose contributions to the assembly process have begun to be elucidated. Here, we review current knowledge regarding the mechanisms by which these core characters function in an orderly fashion to assemble the centriole. In particular, we discuss how having the correct proteins at the right place and at the right time is critical to first scaffold, then initiate, and finally execute the centriole assembly process, thus underscoring fundamental principles governing organelle biogenesis.

The Cep63-Cep152 complex located at the mother centriole recruits Plk4 to initiate the centriole biogenesis. How the complex is targeted to mother centrioles, however, is unclear. In this study, we show that Cep57 and its paralog, Cep57l1, colocalize with Cep63 and Cep152 at the proximal end of mother centrioles in both cycling cells and multiciliated cells undergoing centriole amplification. Both Cep57 and Cep57l1 bind to the centrosomal targeting region of Cep63. The depletion of both proteins, but not either one, blocks the loading of Cep63-Cep152 complex to mother centrioles and consequently prevents centriole duplication. We propose that Cep57 and Cep57l1 function redundantly to ensure the recruitment of the Cep63-Cep152 complex to the mother centrioles for procentriole formation.

Centriole duplication begins with the assembly of a pre-procentriole at a single site on a mother centriole and proceeds with the hierarchical recruitment of a conserved set of proteins, including Polo-like kinase 4 (Plk4)/ZYG-1, Ana2/SAS-5/STIL, and the cartwheel protein Sas6. During assembly, Ana2/STIL stimulates Plk4 kinase activity, and in turn, Ana2/STIL’s C-terminus is phosphorylated, allowing it to bind and recruit Sas6. The assembly steps immediately preceding Sas6-loading appear clear, but the mechanism underlying the upstream pre-procentriole recruitment of Ana2/STIL is not. In contrast to proposed models of Ana2/STIL recruitment, we recently showed that Drosophila Ana2 targets procentrioles independent of Plk4-binding. Instead, Ana2 recruitment requires Plk4 phosphorylation of Ana2’s N-terminus, but the mechanism explaining this process is unknown. Here, we show that the amyloid-like domain of Sas4, a centriole surface protein, binds Plk4 and Ana2, and facilitates phosphorylation of Ana2’s N-terminus which increases Ana2’s affinity for Sas4. Consequently, Ana2 accumulates at the procentriole to induce daughter centriole assembly.

Centrioles play critical roles in organizing the assembly of the mitotic spindle and templating the formation of primary cilia. Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Although significant progress has been made in understanding centriole composition, we have limited knowledge of how PLK4 activity controls specific steps in centriole formation. Here, we show that PLK4 phosphorylates its centriole substrate STIL on a conserved site, S428, to promote STIL binding to CPAP. This phospho-dependent binding interaction is conserved in Drosophila and facilitates the stable incorporation of both STIL and CPAP into the centriole. We propose that procentriole assembly requires PLK4 to phosphorylate STIL in two different regions: phosphorylation of residues in the STAN motif allow STIL to bind SAS6 and initiate cartwheel assembly, while phosphorylation of S428 promotes the binding of STIL to CPAP, linking the cartwheel to microtubules of the centriole wall., eLife digest A cell’s DNA is the chemical instruction manual for everything it does. Each cell in our bodies contains over two meters of DNA, which is divided into 46 packages of information called chromosomes. When the body needs to make more cells, for example during growth or repair, existing cells divide in two in order to replicate themselves. This means that they also need to copy all of their DNA and then deliver identical sets of chromosomes to each new cell. Animal cells use structures called centrioles to help them divide their sets of chromosomes accurately. When cells are about to divide, they make a new set of centrioles by assembling a variety of proteins. This assembly process must be carefully controlled; if too many or too few centrioles are built, cell division errors can occur that lead to the generation of new cells with abnormal numbers of chromosomes. The enzyme PLK4 helps to assemble centrioles, but its exact role in the construction process has remained largely unknown. For example, how it might modify different components of the centriole, and why this matters, is poorly understood. By performing cell biological and biochemical experiments using human cells, Moyer and Holland show that PLK4 interacts with a protein called STIL that is found in the central part of the centriole. The modification of STIL at a specific location by PLK4 was needed to link it to another protein in the outer wall of the centriole, and was also necessary for the cells to build new centrioles. Cells in which PLK4 was unable to modify STIL had too few centrioles when they were beginning to divide. Testing the activity of PLK4 in fruit flies revealed that it plays a similar role as in human cells. This suggests that the modification of STIL by PLK4 is important for normal cell division across different species. The results presented by Moyer and Holland help us to understand how dividing cells build the complex machinery that enables them to pass on their genetic material accurately. Future work that builds on these findings could provide insight into human diseases, such as brain development disorders and cancer, where centrioles are either defective or present in the wrong number.

Centrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. How the core components cooperate to achieve high fidelity in centriole duplication remains poorly understood. By live-cell imaging of endogenously tagged proteins in human cells throughout the entire cell cycle, we quantitatively tracked the dynamics of the critical duplication factors: Plk4, STIL and HsSAS6. Centriolar Plk4 peaks and then starts decreasing during the late G1 phase, which coincides with the accumulation of STIL at centrioles. Shortly thereafter, the HsSAS6 level increases steeply at the procentriole assembly site. We also show that both STIL and HsSAS6 are necessary for attenuating Plk4 levels. Furthermore, our mathematical modeling and simulation suggest that the STIL-HsSAS6 complex in the cartwheel has a negative feedback effect on centriolar Plk4. Combined, these findings illustrate how the dynamic behavior of and interactions between critical duplication factors coordinate the centriole-duplication process. This article has an associated First Person interview with the first author of the paper., Summary: The findings in this study illustrate how the dynamic behavior and interactions between critical duplication factors coordinate the centriole-duplication process in human cells, based on live-cell imaging of endogenously tagged proteins and mathematical modeling.

The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle – from assembly to disappearance – focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.

At the onset of procentriole formation, a structure called the cartwheel is formed adjacent to the pre-existing centriole. SAS-6 proteins are thought to constitute the hub of the cartwheel structure. However, the exact function of the cartwheel in the process of centriole formation has not been well characterized. In this study, we focused on the functions of human SAS-6 (HsSAS-6, also known as SASS6). By using an in vitro reconstitution system with recombinant HsSAS-6, we first observed its conserved molecular property of forming the central part of the cartwheel structure. Furthermore, we uncovered critical functions of HsSAS-6 by using a combination of an auxin-inducible HsSAS-6-degron (AID) system and super-resolution microscopy in human cells. Our results demonstrate that the HsSAS-6 is required not only for the initiation of centriole formation, but also for the stabilization of centriole intermediates. Moreover, after procentriole formation, HsSAS-6 is necessary for limiting Plk4 accumulation at the centrioles and thereby suppressing the formation of initiation sites that would otherwise promote the development of extra procentrioles. Overall, these findings illustrate the conserved and fundamental functions of the cartwheel in centriole duplication.

Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in cancer and was shown to trigger tumorigenesis in mice. However, the incidence, consequence and origins of this abnormality are poorly understood. Here, we screened the NCI-60 panel of human cancer cell lines to systematically analyse centriole number and structure. Our screen shows that centriole amplification is widespread in cancer cell lines and highly prevalent in aggressive breast carcinomas. Moreover, we identify another recurrent feature of cancer cells: centriole size deregulation. Further experiments demonstrate that severe centriole over-elongation can promote amplification through both centriole fragmentation and ectopic procentriole formation. Furthermore, we show that overly long centrioles form over-active centrosomes that nucleate more microtubules, a known cause of invasiveness, and perturb chromosome segregation. Our screen establishes centriole amplification and size deregulation as recurrent features of cancer cells and identifies novel causes and consequences of those abnormalities., Cancer cells are characterised by abnormalities in the number of centrosomes and this phenotype is linked with tumorigenesis. Here the authors report centriole length deregulation in a subset of cancer cell lines and suggest a link with subsequent alterations in centriole numbers and chromosomal instability.

Mammalian epithelial cells use a pair of mother centrioles (MCs) and numerous deuterosomes as platforms for efficient basal body assembly during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to either arise spontaneously followed by maturation into larger ones with increased procentriole-producing capacity or be assembled solely on the young MC, nucleate procentrioles under the MC’s guidance, and released as procentriole-occupied "halos". Here we show that both MCs are dispensable for deuterosome formation in multiciliate cells. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm were initially procentriole-free and then grew into halos. More importantly, eliminating the young MC or both MCs in proliferating precursor cells through shRNA-mediated depletion of Plk4, a kinase essential to procentriole assembly, did not abolish deuterosome formation when these cells were induced to differentiate into mEPCs. The average deuterosome numbers per cell only reduced by 21% as compared to control mEPCs. Therefore, MC is not essential to the assembly of both deuterosomes and deuterosome-mediated procentrioles.

The centrosome is the organizing center of microtubules in the cell, the basis for the origin of cilia and flagella and a site for the concentration of a regulatory proteins multitude. The centrosome comprises two centrioles surrounded by pericentriolar material. Centrioles in the cells of different organisms can contain nine triplets, doublets or singlets of microtubules. Here, we show that in somatic cells of male wasp larvae Anisopteromalus calandrae, centrioles do not contain microtubules and are composed of nine electron-dense prongs, which together form a cogwheel structure. These microtubule-free centrioles can be the platform for procentriole formation and form microtubule-free cilia-like structures. In nymph and imago cells centrioles have a microtubule triplet structure. Our study describes how centriole structure differs in a development-stage-dependent and a cell-type-dependent manner. The discovery of a centriole without microtubules casts a new light on the centriole formation process and the evolution of this organelle., Summary: Centrioles without microtubules in larvae cells of parasitoid wasps duplicate and form primary cilia without microtubules, in nymph and imago cells centrioles acquire a ‘normal’ structure with nine microtubules triplets.

Single-particle reconstruction (SPR) from electron microscopy images is widely used in structural biology, but lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and co-aligns multiple proteins from 2D super-resolution fluorescence images. We demonstrate our method by generating multi-color 3D reconstructions of several proteins within the human centriole and procentriole, revealing their relative locations, dimensions and orientations.

Centrioles are conserved microtubule-based organelles that form the core of the centrosome and act as templates for the formation of cilia and flagella. Centrioles have important roles in most microtubule-related processes, including motility, cell division and cell signalling. To coordinate these diverse cellular processes, centriole number must be tightly controlled. In cycling cells, one new centriole is formed next to each pre-existing centriole in every cell cycle. Advances in imaging, proteomics, structural biology and genome editing have revealed new insights into centriole biogenesis, how centriole numbers are controlled and how alterations in these processes contribute to diseases such as cancer and neurodevelopmental disorders. Moreover, recent work has uncovered the existence of surveillance pathways that limit the proliferation of cells with numerical centriole aberrations. Owing to this progress, we now have a better understanding of the molecular mechanisms governing centriole biogenesis, opening up new possibilities for targeting these pathways in the context of human disease.

SummarySymmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the “ring”, to a unique PLK4 focus, the “spot”, that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals,in situdegradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

Centriole duplication is tightly regulated throughout the cell cycle to ensure one duplication event per centriole. McLamarrah et al. show that a stepwise pattern of Ana2 phosphorylation by Plk4 facilitates proper centriole duplication., Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4–Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

Male gametogenesis in insects is unusual in that the centrioles do not duplicate during the second meiosis and the differentiating spermatids inherit only one centriole. Here it is showed that a distinct procentriole is assembled close to the proximal region of the centriole in early S13 spermatids at the onion stage, confirming previous reports of a proximal centriole-like structure at the proximal end of the spermatid centriole. However, the procentrioles of Drosophila spermatids do not behave like true procentrioles, but their development is blocked at an early stage before the assembly of a complete A-tubule set. Therefore, they may represent early frozen stages of procentriole assembly that do not develop further and eventually disappear in late spermatids.

The deposited article is a post-print version and has been submitted to peer review. This publication hasn't any creative commons license associated. The deposited article version contains attached the supplementary materials within the pdf. Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions. Fundação Portuguesa para a Ciência e Tecnologia grants: (SFRH/BPD/87479/2012, PTDC/BBB-BEP/1724/2012, HMSP-CT/SAU-ICT/0075/2009, PTDC/SAU-OBD/105616/2008, EXPL/BIM-ONC/0830/2013, PTDC/BBB-BEP/1724/2012); EMBO installation grant; ERC starting grant: (PFE-GI-UE-ERC-2010-StG-261344). info:eu-repo/semantics/publishedVersion

Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL) and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication.

Binding of STIL activates Plk4, and the subsequent phosphorylation of STIL by Plk4 primes the binding of STIL to SAS6 to promote centriole assembly., Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle–regulated accumulation of STIL.

The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL (respective nomenclature of worms/flies/humans). Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. Here we show that in Drosophila , Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When we over-ride the auto-destruction of Plk4, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. We now show that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region we term the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6. We discuss these findings in light of the multiple Plk4 phosphorylation sites on Ana2.

We studied spermatogenesis and spermiogenesis in Adalia decempunctata (L), a beetle of the Coccinellidae family. The spermatocyte exhibits two centrioles which elongate to form a pair of primary cilia. A novel structure, appearing in cross sections as a dense droplet, is observed near the long centriole during spermiogenesis, and is soon accompanied by a procentriole (PCL). PCL structure consists of singlet microtubules, a central tubule and an incomplete cartwheel. The PCL persists until the end of spermiogenesis, when it vanishes together with the dense droplet. The sperm has an exceptionally long basal body and the nucleus is disposed parallel to the flagellar components, a peculiar trait shared by other species of the coccinellid group. The presence of a procentriole suggested by the use of antibodies is discussed.

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly., Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles

Summary: The number of centrioles is tightly controlled to ensure bipolar spindle assembly, which is a prerequisite to maintain genome integrity. However, our understanding of the fundamental principle that governs the formation of a single procentriole per parental centriole is incomplete. Here, we show that the local restriction of Plk4, a master regulator of the procentriole formation, is achieved by a bimodal interaction of STIL with Plk4. We demonstrate that the conserved short coiled-coil region of STIL binds to and protects Plk4 from protein degradation at the site of procentriole formation. On the other hand, the conserved C-terminal region of STIL named truncated in microcephaly (TIM) domain promotes autophosphorylation and degradation of adjacent Plk4 by the direct interaction. Thus, we propose that positive and negative regulation based on the bimodal binding of Plk4 and STIL ensures the formation of a single procentriole per parental centriole. : Ohta et al. show that Plk4 asymmetrically localizes around mother centrioles before the onset of procentriole formation. Furthermore, they reveal that bimodal binding of STIL to Plk4 restricts Plk4 localization at a single site and thus ensures formation of a single procentriole per mother centriole. Keywords: cell division, centrosome, centriole duplication, Plk4, STIL

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897–1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP897−1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP897−1338 (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis., This work was supported by the Comunidad de Madrid through grant CAM (S2010/BMD- 2305) and the Spanish Ministry of Economy and Competitiveness through Grants AIC-A-2011-0638 and BIO2013-44647-R.

Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. Here we showed that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal structure analyses revealed that Cep192 - and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152-peptide bound to the CPB markedly better than the Cep192-peptide and effectively snatched the CPB away from a preformed CPB–Cep192-peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.

HsSAS-6 homodimers are present in the cytoplasm and assemble into ninefold symmetrical arrays at centrosomes, thus initiating procentriole formation., SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.

Centrioles are cylindrical structures that are usually composed of nine triplets of microtubules (MTs) organized around a cartwheel-shaped structure. Recent studies have proposed a structural model of the SAS-6-based cartwheel, yet we do not know the molecular detail of how the cartwheel participates in centriolar MT assembly. In this study, we demonstrate that the human microcephaly protein, CEP135, directly interacts with hSAS-6 via its carboxyl-terminus and with MTs via its amino-terminus. Unexpectedly, CEP135 also interacts with another microcephaly protein CPAP via its amino terminal domain. Depletion of CEP135 not only perturbed the centriolar localization of CPAP, but also blocked CPAP-induced centriole elongation. Furthermore, CEP135 depletion led to abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. Overexpression of a CEP135 mutant lacking the proper interaction with hSAS-6 had a dominant-negative effect on centriole assembly. We propose that CEP135 may serve as a linker protein that directly connects the central hub protein, hSAS-6, to the outer MTs, and suggest that this interaction stabilizes the proper cartwheel structure for further CPAP-mediated centriole elongation.

SummaryThe centrosome functions as the main microtubule-organizing center of animal cells and is crucial for several fundamental cellular processes [1]. Abnormalities in centrosome number and composition correlate with tumor progression [2, 3] and other diseases [4–6]. Although proteomic studies have identified many centrosomal proteins, their interactions are incompletely characterized [7, 8]. The lack of information on the precise localization and interaction partners for many centrosomal proteins precludes comprehensive understanding of centrosome biology. Here, we utilize a combination of selective chemical crosslinking and superresolution microscopy to reveal novel functional interactions among a set of 31 centrosomal proteins. We reveal that Cep57, Cep63, and Cep152 are parts of a ring-like complex localizing around the proximal end of centrioles. Furthermore, we identify that STIL, together with HsSAS-6, resides at the proximal end of the procentriole, where the cartwheel is located. Our studies also reveal that the known interactors Cep152 and Plk4 reside in two separable structures, suggesting that the kinase Plk4 contacts its substrate Cep152 only transiently, at the centrosome or within the cytoplasm. Our findings provide novel insights into protein interactions critical for centrosome biology and establish a toolbox for future studies of centrosomal proteins.