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Supernumerary centrioles are frequently observed in diverse types of cancer cells. In this study, we investigated the mechanism underlying the generation of supernumerary centrioles during the M phase. We generated the TP53;PCNT;CEP215 triple knockout (KO) cells and determined the configurations of the centriole during the cell cycle. The triple KO cells exhibited a precocious separation of centrioles and unscheduled centriole assembly in the M phase. Supernumerary centrioles in the triple KO cells were present throughout the cell cycle; however, among all the centrioles, only two maintained an intact composition, including CEP135, CEP192, CEP295 and CEP152. Intact centrioles were formed during the S phase and the rest of the centrioles may be generated during the M phase. M-phase-assembled centrioles lacked the ability to organize microtubules in the interphase; however, a fraction of them may acquire pericentriolar material to organize microtubules during the M phase. Taken together, our work reveals the heterogeneity of the supernumerary centrioles in the triple KO cells. .

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 (MT) cytoskeleton, centrosome, and centriolar satellites. SON is required for the proper splicing and expression of CEP131, which encodes a major centriolar satellite protein and is required to organize the trafficking and MT network around the centrosomes. This study highlights the importance of the distinct MT trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis.

The G-quadruplexes (G4s) are a class of DNA secondary structures with guanine rich DNA sequences that can fold into four stranded non-canonical structures. At the genomic level, their pivotal role is well established in DNA replication, telomerase functions, constitution of topologically associating domains, and the regulation of gene expression. Genome instability mediated by altered G4 formation and assembly has been associated with multiple disorders including cancers and neurodegenerative disorders. Multiple tools have also been developed to predict the potential G4 regions in genomes and the whole genome G4 maps are also being derived through sequencing approaches. Enrichment of G4s in the cis-regulatory elements of genes associated with tumorigenesis has accelerated the quest for identification of G4-DNA binding ligands (G4DBLs) that can selectively bind and regulate the expression of such specific genes. In this context, the analysis of G4DBL responsive transcriptome in diverse cancer cell lines is inevitable for assessment of the specificity of novel G4DBLs. Towards this, we assembled the transcripts differentially regulated by different G4DBLs and have also identified a core set of genes regulated in diverse cancer cell lines in response to 3 or more of these ligands. With the mode of action of G4DBLs towards topology shifts, folding, or disruption of G4 structure being currently visualized, we believe that this dataset will serve as a platform for assembly of G4DBL responsive transcriptome for comparative analysis of G4DBLs in multiple cancer cells based on the expression of specific cis-regulatory G4 associated genes in the future. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12079-021-00637-z.

The centrosome plays an essential role in maintaining genetic stability, ciliogenesis and cell polarisation. The core of the centrosome is made up of two centrioles that duplicate precisely once during every cell cycle to generate two centrosomes that are required for bipolar spindle assembly and chromosome segregation. Abundance of centriole proteins at optimal levels and their recruitment to the centrosome are tightly regulated in time and space in order to restrict aberrant duplication of centrioles, a phenomenon that is observed in many cancers. Recent advances have conclusively shown that dedicated ubiquitin ligase-dependent protein degradation machineries are involved in governing centriole duplication. These studies revealed intricate mechanistic insights into how the ubiquitin ligases target different centriole proteins. In certain cases, a specific ubiquitin ligase targets a number of substrate proteins that co-regulate centriole assembly, prompting the possibility that substrate-targeting occurs during formation of the sub-centriolar structures. There are also instances where a specific centriole duplication protein is targeted by several ubiquitin ligases at different stages of the cell cycle, suggesting synchronised actions. Recent evidence also indicated a direct association of E3 ubiquitin ligase with the centrioles, supporting the notion that substrate-targeting occurs in the organelle itself. In this review, we highlight these advances by underlining the mechanisms of how different ubiquitin ligase machineries control centriole duplication and discuss our views on their coordination.

Centrosomes were first described by Edouard Van Beneden and named and linked to chromosome segregation by Theodor Boveri around 1870. In the 1960-1980s, electron microscopy studies have revealed the remarkable ultrastructure of a centriole -- a nine-fold symmetrical microtubular assembly that resides within a centrosome and organizes it. Less than two decades ago, proteomics and genomic screens conducted in multiple species identified hundreds of centriole and centrosome core proteins and revealed the evolutionarily conserved nature of the centriole assembly pathway. And now, super resolution microscopy approaches and improvements in cryo-tomography are bringing an unparalleled nanoscale-detailed picture of the centriole and centrosome architecture. In this chapter, we summarize the current knowledge about the architecture of human centrioles. We discuss the structured organization of centrosome components in interphase, focusing on localization/function relationship. We discuss the process of centrosome maturation and mitotic spindle pole assembly in centriolar and acentriolar cells, emphasizing recent literature.

The centrosome, composed of two centrioles surrounded by pericentriolar material, is the cell’s central microtubule-organizing center. Centrosome duplication is coupled with the cell cycle such that centrosomes duplicate once in S phase. Loss of such coupling produces supernumerary centrosomes, a condition called centrosome amplification (CA). CA promotes cell invasion and chromosome instability, two hallmarks of cancer. We examined the contribution of centriole overduplication to CA and the consequences for genomic stability in breast cancer cells. CEP135, a centriole assembly protein, is dysregulated in some breast cancers. We previously identified a short isoform of CEP135, CEP135mini, that represses centriole duplication. Here, we show that the relative level of full-length CEP135 (CEP135full) to CEP135mini (the CEP135full:mini ratio) is increased in breast cancer cell lines with high CA. Inducing expression of CEP135full in breast cancer cells increases the frequency of CA, multipolar spindles, anaphase-lagging chromosomes, and micronuclei. Conversely, inducing expression of CEP135mini reduces centrosome number. The differential expression of the CEP135 isoforms in vivo is generated by alternative polyadenylation. Directed genetic mutations near the CEP135mini alternative polyadenylation signal reduces the CEP135full:mini ratio and decreases CA. We conclude that dysregulation of CEP135 isoforms promotes centriole overduplication and contributes to chromosome segregation errors in breast cancer cells.

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole “targeting-factor.” In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4’s kinase domain. Thus, Asl-A’s phosphorylation state determines which of Asl-A’s two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

Centriole-to-centrosome conversion (CCC) safeguards centriole homeostasis by coupling centriole duplication with segregation, and is essential for stabilization of mature vertebrate centrioles naturally devoid of the geometric scaffold or the cartwheel. Here we identified PPP1R35, a putative regulator of the protein phosphatase PP1, as a novel centriolar protein required for CCC. We found that PPP1R35 is enriched at newborn daughter centrioles in S or G2 phase. In the absence of PPP1R35, centriole assembly initiates normally in S phase, but none of the nascent centrioles can form active centrosomes or recruit CEP295, an essential factor for CCC. Instead, all PPP1R35-null centrioles, although stable during their birth in interphase, become disintegrated after mitosis upon cartwheel removal. Surprisingly, we found that neither the centriolar localization nor the function of PPP1R35 in CCC requires the putative PP1-interacting motif. PPP1R35 is thus acting upstream of CEP295 to induce CCC for proper centriole maintenance.

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.

Centrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes assemble centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, we show that, during spermatogenesis of the bryophyte Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. Interestingly, we observe that these centrioles are twisted in opposite orientations. Microtubules emanate from the bicentrioles, which localize to the spindle poles during cell division. After their separation, the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two motile cilia are assembled that appear to alternate between different motility patterns. We further show that centriolar components SAS6, Bld10, and POC1, which are conserved across eukaryotes, are expressed during spermatogenesis and required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis, while driven by conserved molecular modules, is more diverse than previously thought.

Centrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes form centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, combining electron microscopy and tomography, we show that during spermatogenesis of the moss Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. We observe that microtubules emanate from those bicentrioles, which localize to the spindle poles during cell division. Thereafter, each bicentriole breaks apart, and the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two cilia are assembled which are capable of beating asynchronously. We further show that conserved cartwheel and centriole wall components, SAS6, BLD10 and POC1 are expressed during spermatogenesis and are required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis is more diverse than previously thought and that conserved molecular modules underlie diversification of this essential pathway.

/SummaryCentrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes form centriolesde novothrough uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, combining electron microscopy and tomography, we show that during spermatogenesis of the mossPhyscomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. We observe that microtubules emanate from those bicentrioles, which localize to the spindle poles during cell division. Thereafter, each bicentriole breaks apart, and the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two cilia are assembled which are capable of beating asynchronously. We further show that conserved cartwheel and centriole wall components, SAS6, BLD10 and POC1 are expressed during spermatogenesis and are required for thisde novobiogenesis pathway. Our work supports a scenario where centriole biogenesis is more diverse than previously thought and that conserved molecular modules underlie diversification of this essential pathway.

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease characterized by impaired growth and increased tumorigenesis, whose cellular etiology is poorly understood. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterized these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We established that an atypical de novo assembly pathway is notably involved in forming Cenpas, which can nevertheless trigger further centriole assembly and act as MTOCs. We found also that Cenpas are present and act similarly in Mulibrey patient cells. Through correlative light electron microscopy, we uncovered that Cenpas correspond to centriole related structures and elongated electron-dense structures with stripes. Importantly, we established that TRIM37 regulates the stability and solubility of the centriolar protein Centrobin. Our findings suggest that elongated Centrobin assemblies are a major constituent of the striped electron dense structures. Furthermore, we established that Cenpas formation upon TRIM37 depletion requires PLK4 activity, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents the formation of Cenpas that would otherwise threaten genome integrity, including possibly in Mulibrey patients.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. In the proximal region of the centriole, nine microtubule triplets attach to each other via A-C linkers and encircle a central cartwheel structure, which directs the early events of centriole assembly. While the crucial role of the proximal region in centriole biogenesis has been well documented in many species, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, including humans, we report on the architectural diversity of the centriolar proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub, previously reported to have an 8.5 nm periodicity in Trichonympha, is constructed from a stack of paired rings with an average periodicity of ∼4 nm. In all four examined species, cartwheel inner densities are found inside the hub’s ring-pairs. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring-pairs with 6 radial spokes emanating and merging into a single bundle that connects to the triplet microtubule via the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through a flexible triplet-base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

In this Primer, Nabais et al. discuss the evolution of the structure and function of centrioles and basal bodies, describe conserved centriole assembly features and the diversity in centriole architecture across eukaryotes, and highlight important outstanding evolutionary questions concerning centriole assembly.

Nabais et al. use an egg explant system overexpressing Plk4 to study the spatiotemporal and biochemical regulation of de novo centriole assembly. They show that the onset and kinetics of biogenesis depend on Plk4 concentration, requiring the matrix that surrounds centrioles., Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. Herein, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, our results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and found that proteins of the pericentriolar material (PCM), and in particular γ-tubulin, promote biogenesis, likely by locally concentrating critical components.

Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins [Plk4, Ana2 (the homologue of mammalian STIL), Sas-6, Sas-4 (mammalian CPAP) or Asl (mammalian Cep152)] leads to the absence of centrioles. Here, we use Sas-6 and Ana2 particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs or embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, whereas Asl helps to initiate SAP assembly but is not required for SAP growth. Although not centrioles, SAPs recruit and organise many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl to efficiently recruit pericentriolar material (PCM), but Spd-2 (the homologue of mammalian Cep192) can promote some PCM assembly independently of Asl. These observations provide new insights into the pathways of centriole and centrosome assembly.

Centrioles are microtubule-based organelles that organize the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a stepwise process dependent notably on the centriolar protein SAS-5 in Caenorhabditis elegans. SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules. Microtubule binding requires SAS-5 oligomerization and a disordered protein segment that overlaps with the SAS-4 binding site. Combined in vitro and in vivo analysis of select mutants reveals that the SAS-5–microtubule interaction facilitates centriole assembly in C. elegans embryos. Our findings lead us to propose that the interdependence of SAS-5 oligomerization and microtubule binding reflects an avidity mechanism, which also strengthens SAS-5 associations with other centriole components and, thus, promotes organelle assembly.

POC1A encodes a protein with a role in centriole assembly and stability, and in ciliogenesis. Biallelic loss-of-function mutations affecting POC1A cause SOFT syndrome, an ultra-rare condition characterized by short stature, onychodysplasia, facial dysmorphism and hypotrichosis. Using exome sequencing, we identified a homozygous frameshift mutation (c.1047_1048dupC; p.G337Rfs*25) in a patient presenting with short stature, facial hirsutism, alopecia, dyslipidemia and extreme insulin resistance. The truncating variant affected exon 10, which is retained in only two of the three POC1A-mature RNAs, due to alternative processing of the transcript. Clinical discrepancies with SOFT syndrome support the hypothesis that POC1A mutations affecting exon 10 are associated with a distinct condition, corroborating a previous hypothesis based on a similar case. Furthermore, this report provides an additional example of a genetic condition presenting with clinical heterogeneity due to alternative transcript processing. In conclusion, POC1A mutations in exon 10 should be taken into account in patients with extreme insulin resistance and short stature.

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.

Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.

Centrioles are subcellular organelles that were present in the last eukaryotic common ancestor, where the centriole's ancestral role was to form cilia. Centrioles have maintained a remarkably conserved structure in eukaryotes that have cilia, while groups that lack cilia have lost their centrioles, highlighting the structure-function relationship that exists between the centriole and the cilium. In contrast, animal sperm cells, a ciliated cell, exhibit remarkable structural diversity in the centriole. Understanding how this structural diversity evolved may provide insight into centriole assembly and function, as well as their unique role in sperm. Here, we apply concepts used in the study of the evolution of animal morphology to gain insight into the evolution of centriole structure. We propose that centrioles with an atypical structure form because of changes in the timing of centriole assembly events, which can be described as centriolar "heterochrony." Atypical centrioles of insects and mammals appear to have evolved through different types of heterochrony. Here, we discuss two particular types of heterochrony: neoteny and hypermorphosis. The centriole assembly of insect sperm cells exhibits the retention of "juvenile" centriole structure, which can be described as centriolar "neoteny." Mammalian sperm cells have an extended centriole assembly program through the addition of novel steps such as centrosome reduction and centriole remodeling to form atypical centrioles, a form of centriole "hypermorphosis." Overall, centriole heterochrony appears to be a common mechanism for the development of the atypical centriole during the evolution of centriole assembly of various animals' sperm.

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.

Centrosomes are tiny yet complex cytoplasmic structures that perform a variety of roles related to their ability to act as microtubule-organizing centers. Like the genome, centrosomes are single copy structures that undergo a precise semi-conservative replication once each cell cycle. Precise replication of the centrosome is essential for genome integrity, because the duplicated centrosomes will serve as the poles of a bipolar mitotic spindle, and any number of centrosomes other than two will lead to an aberrant spindle that mis-segregates chromosomes. Indeed, excess centrosomes are observed in a variety of human tumors where they generate abnormal spindles in situ that are thought to participate in tumorigenesis by driving genomic instability. At the heart of the centrosome is a pair of centrioles, and at the heart of centrosome duplication is the replication of this centriole pair. Centriole replication proceeds through a complex macromolecular assembly process. However, while centrosomes may contain as many as 500 proteins, only a handful of proteins have been shown to be essential for centriole replication. Our observations suggest that centriole replication is a modular, bottom-up process that we envision akin to building a house; the proper site of assembly is identified, a foundation is assembled at that site, and subsequent modules are added on top of the foundation. Here, we discuss the data underlying our view of modularity in the centriole assembly process, and suggest that non-essential centriole assembly factors take on greater importance in cancer cells due to their function in coordination between centriole modules, using the Monopolar spindles 1 protein kinase and its substrate Centrin 2 to illustrate our model.

SUMMARY PLK4 is the major kinase driving centriole duplication. Duplication occurs only once per cell cycle, forming one new (or daughter) centriole that is tightly engaged to the preexisting (or mother) centriole. Centriole engagement is known to block the reduplication of mother centrioles, but the molecular identity responsible for the block remains unclear. Here, we show that the centriolar cartwheel, the geometric scaffold for centriole assembly, forms the identity of daughter centrioles essential for the block, ceasing further duplication of the mother centriole to which it is engaged. To ensure a steady block, we found that the cartwheel requires constant maintenance by PLK4 through phosphorylation of the same substrate that drives centriole assembly, revealing a parsimonious control in which “assembly” and “block for new assembly” are linked through the same catalytic reaction to achieve homeostasis. Our results support a recently deduced model that the cartwheel-bound PLK4 directly suppresses centriole reduplication., Graphical Abstract

Cytokine production is a necessary event in the immune response during inflammation and is associated with mortality during sepsis, autoimmune disorders, cancer, and diabetes. Stress-activated MAP kinase signaling cascades that mediate cytokine synthesis are well established. However, the downstream fate of cytokines before they are secreted remains elusive. We report that pro-inflammatory stimuli lead to recruitment of pericentriolar material, specifically pericentrin and γ-tubulin, to the centrosome. This is accompanied by enhanced microtubule nucleation and enrichment of the recycling endosome component FIP3, all of which are hallmarks of centrosome maturation during mitosis. Intriguingly, centrosome maturation occurs during interphase in an MLK-dependent manner, independent of the classic mitotic kinase, Plk1. Centrosome disruption by chemical prevention of centriole assembly or genetic ablation of pericentrin attenuated interleukin-6, interleukin-10, and MCP1 secretion, suggesting that the centrosome is critical for cytokine production. Our results reveal a function of the centrosome in innate immunity.

Centrioles are eukaryotic organelles whose number and position are critical for cilia formation and mitosis. Many cell types assemble new centrioles next to existing ones ("templated" or mentored assembly). Under certain conditions, centrioles also form without pre-existing centrioles (de novo). The synchronous differentiation of Naegleria amoebae to flagellates represents a unique opportunity to study centriole assembly, as nearly 100% of the population transitions from having no centrioles to having two within minutes. Here, we find that Naegleria forms its first centriole de novo, immediately followed by mentored assembly of the second. We also find both de novo and mentored assembly distributed among all major eukaryote lineages. We therefore propose that both modes are ancestral and have been conserved because they serve complementary roles, with de novo assembly as the default when no pre-existing centriole is available, and mentored assembly allowing precise regulation of number, timing, and location of centriole assembly.

Centrioles play critical roles in the organization of microtubule-based structures, from the mitotic spindle to cilia and flagella. In order to properly execute their various functions, centrioles are subjected to stringent copy number control. Central to this control mechanism is a precise duplication event that takes place during S phase of the cell cycle and involves the assembly of a single daughter centriole in association with each mother centriole . Recent studies have revealed that posttranslational control of the master regulator Plk4/ZYG-1 kinase and its downstream effector SAS-6 is key to ensuring production of a single daughter centriole. In contrast, relatively little is known about how centriole duplication is regulated at a transcriptional level. Here we show that the transcription factor complex EFL-1-DPL-1 both positively and negatively controls centriole duplication in the Caenorhabditis elegans embryo. Specifically, we find that down regulation of EFL-1-DPL-1 can restore centriole duplication in a zyg-1 hypomorphic mutant and that suppression of the zyg-1 mutant phenotype is accompanied by an increase in SAS-6 protein levels. Further, we find evidence that EFL-1-DPL-1 promotes the transcription of zyg-1 and other centriole duplication genes. Our results provide evidence that in a single tissue type, EFL-1-DPL-1 sets the balance between positive and negative regulators of centriole assembly and thus may be part of a homeostatic mechanism that governs centriole assembly.

Cetn3 inhibits Mps1 kinase activity in vitro and at centrosomes by blocking activating autophosphorylation and can prevent Mps1 from phosphorylating Cetn2 even when Mps1 is present at 10-fold molar excess. Cetn3 also prevents incorporation of Cetn2 into centrioles, but mimicking phosphorylation of Cetn2 bypasses the inhibitory effects of Cetn3., Centrins are a family of small, calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified centrin 2 and centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, although Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole overproduction, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, whereas depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr-676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes.

Centriole duplication is coordinated such that a single round of duplication occurs during each cell cycle. Disruption of this synchrony causes defects including supernumerary centrosomes in cancer and perturbed ciliary signaling [1-5]. To preserve the normal number of centrioles, the level, localization, and post-translational modification of centriole proteins is regulated so that, when centriole protein expression and/or activity are increased, centrioles self-assemble. Assembly is initiated by the formation of the cartwheel structure that comprises the base of centrioles [6-11]. SAS-6 constitutes the cartwheel, and SAS-6 levels remain low until centriole assembly is initiated at S phase onset [3, 12, 13]. CEP135 physically links to SAS-6 near the site of microtubule nucleation and binds to CPAP for triplet microtubule formation [13, 14]. We identify two distinct protein isoforms of CEP135 that antagonize each other to modulate centriole duplication: full-length CEP135 (CEP135(full)) promotes new assembly, whereas a short isoform, CEP135(mini), represses it. CEP135(mini) represses centriole duplication by limiting the centriolar localization of CEP135(full) binding proteins (SAS-6 and CPAP) and the pericentriolar localization of γ-tubulin. The CEP135 isoforms exhibit distinct and complementary centrosomal localization during the cell cycle. CEP135(mini) protein decreases from centrosomes upon anaphase onset. We suggest that the decrease in CEP135(mini) from centrosomes promotes centriole assembly. The repression of centriole duplication by a splice isoform of a protein that normally promotes it serves as a novel mechanism to limit centriole duplication.

Theodor Boveri is considered as the “father” of centrosome biology. Boveri’s fundamental findings have laid the groundwork for decades of research on centrosomes. Here, we briefly review his early work on centrosomes and his first description of the centriole. Mainly focusing on centriole structure, duplication, and centriole assembly factors in C. elegans, we will highlight the role of this model in studying germ line centrosomes in nematodes. Last but not least, we will point to future directions of the C. elegans centrosome field.

Centrosomes are required for faithful chromosome segregation during mitosis. They are composed of a centriole pair that recruits and organizes the microtubule-nucleating pericentriolar material. Centriole duplication is tightly controlled in vivo and aberrations in this process are associated with several human diseases, including cancer and microcephaly. Although factors essential for centriole assembly, such as STIL and PLK4, have been identified, the underlying molecular mechanisms that drive this process are incompletely understood. Combining protein proximity mapping with high-resolution structural methods, we identify CEP85 as a centriole duplication factor that directly interacts with STIL through a highly conserved interaction interface involving a previously uncharacterised domain of STIL. Structure-guided mutational analyses in vivo demonstrate that this interaction is essential for efficient centriolar targeting of STIL, PLK4 activation and faithful daughter centriole assembly. Taken together, our results illuminate a molecular mechanism underpinning the spatiotemporal regulation of the early stages of centriole duplication., Centriole duplication is tightly regulated in vivo, but the underlying molecular mechanisms are incompletely understood. Here the authors use high-resolution structural and imaging methods to show that CEP85 directly interacts with STIL and mediates efficient centriolar targeting of STIL, PLK4 activation and centriole assembly.

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar matrix (PCM). Centrioles regulate matrix assembly. Here we ask whether the matrix also regulates centriole assembly. To define the interaction between the matrix and individual centriole components, we take advantage of a heterologous expression system using fission yeast. Importantly, its centrosome, the spindle pole body (SPB), has matrix but no centrioles. Surprisingly, we observed that the SPB can recruit several animal centriole components. Pcp1/pericentrin, a conserved matrix component that is often upregulated in cancer, recruits a critical centriole constituent, SAS-6. We further show that this novel interaction is conserved and important for centriole biogenesis and elongation in animals. We speculate that the Pcp1/pericentrin-SAS-6 interaction surface was conserved for one billion years of evolution after centriole loss in yeasts, due to its conserved binding to calmodulin. This study reveals an ancestral relationship between pericentrin and the centriole, where both regulate each other assembly, ensuring mutual localisation.

Cycling cells maintain centriole number at precisely two per cell in part by limiting their duplication to S phase under the control of the cell cycle machinery. In contrast, postmitotic multiciliated cells (MCCs) uncouple centriole assembly from cell cycle progression and produce hundreds of centrioles in the absence of DNA replication to serve as basal bodies for motile cilia. Although some cell cycle regulators have previously been implicated in motile ciliogenesis, how the cell cycle machinery is employed to amplify centrioles is unclear. We use transgenic mice and primary airway epithelial cell culture to show that Cdk2, the kinase responsible for the G1 to S phase transition, is also required in MCCs to initiate motile ciliogenesis. While Cdk2 is coupled with cyclins E and A2 during cell division, cyclin A1 is required during ciliogenesis, contributing to an alternative regulatory landscape that facilitates centriole amplification without DNA replication.

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.

Giving an old organelle the old heave-ho Centrioles are ancient cellular organelles that build centrosomes, the major microtubule-organizing centers in animal cells. Duplication of centrioles is tightly controlled to ensure that each dividing cell has precisely two centrosomes. Human cancer cells often have extra centrosomes, which has been hypothesized to confer a proliferative advantage. Wong et al. developed small molecules (centrinones) that allowed them to reversibly “delete” centrioles from cells (see the Perspective by Stearns). Surprisingly, cancer cells continued to divide in the absence of centrosomes, whereas normal cells stopped dividing. Science , this issue p. 1155 ; see also p. 1091

The Asterless N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas its C terminus stabilizes Plk4 during mitosis., Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.

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.

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

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare disease associated with primary infertility; however, ~50% of the genetic alterations associated with MMAF remain unclear. Here, we reported the case of a 30-year-old infertile male from a consanguineous family. Whole-exome sequencing identified a homozygous mutation in the CEP135 gene (c.A1364T:p.D455V), with CEP135 previously reported to play a role in centriole biogenesis and specifically central pair assembly. D455V-mutated proteins formed protein aggregates in the centrosome and the flagella, which might potentially affect the function of centriole assembly. Moreover, intracytoplasmic sperm injection was performed using sperm from this patient; however, pregnancy failed following embryo transfer. This represents the first report of a homozygous mutation of CEP135 associated with MMAF. These results provide researchers and clinicians with a deeper understanding of the gene involved with MMAF and will help predict and assess pregnancy outcomes associated with in vitro fertilization.

Centrioles are microtubule-based cylinders essential for the formation of centrosomes and cilia. A recent study provides a new cell-free assay that reconstitutes the initial structure formed during centriole assembly - the cartwheel - and proposes a new model for its formation and growth.

Centrioles are assembled during S phase and segregated into 2 daughter cells at the end of mitosis. The initiation of centriole assembly is regulated by polo-like kinase 4 (PLK4), the major serine/threonine kinase in centrioles. Despite its importance in centriole duplication, only a few substrates have been identified, and the detailed mechanism of PLK4 has not been fully elucidated. CP110 is a coiled-coil protein that plays roles in centriolar length control and ciliogenesis in mammals. Here, we revealed that PLK4 specifically phosphorylates CP110 at the S98 position. The phospho-resistant CP110 mutant inhibited centriole assembly, whereas the phospho-mimetic CP110 mutant induced centriole assembly, even in PLK4-limited conditions. This finding implies that PLK4 phosphorylation of CP110 is an essential step for centriole assembly. The phospho-mimetic form of CP110 augmented the centrosomal SAS6 level. Based on these results, we propose that the phosphorylated CP110 may be involved in the stabilization of cartwheel SAS6 during centriole assembly.

The centriole/basal body is a eukaryotic organelle that plays essential roles in cell division and signaling. Among five known core centriole proteins, SPD-2/Cep192 is the first recruited to the site of daughter centriole formation and regulates the centriolar localization of the other components in C. elegans and in humans. However, the molecular basis for SPD-2 centriolar localization remains unknown. Here, we describe a new centriole component, the coiled-coil protein SAS-7, as a regulator of centriole duplication, assembly and elongation. Intriguingly, our genetic data suggest that SAS-7 is required for daughter centrioles to become competent for duplication, and for mother centrioles to maintain this competence. We also show that SAS-7 binds SPD-2 and regulates SPD-2 centriolar recruitment, while SAS-7 centriolar localization is SPD-2-independent. Furthermore, pericentriolar material (PCM) formation is abnormal in sas-7 mutants, and the PCM-dependent induction of cell polarity that defines the anterior-posterior body axis frequently fails. We conclude that SAS-7 functions at the earliest step in centriole duplication yet identified and plays important roles in the orchestration of centriole and PCM assembly.

SummaryPlk4 family kinases control centriole assembly. Plk4s target mother centrioles through an interaction between their cryptic polo box (CPB) and acidic regions in the centriolar receptors SPD-2/Cep192 and/or Asterless/Cep152. Here, we report a crystal structure for the CPB of C. elegans ZYG-1, which forms a Z-shaped dimer containing an intermolecular β sheet with an extended basic surface patch. Biochemical and in vivo analysis revealed that electrostatic interactions dock the ZYG-1 CPB basic patch onto the SPD-2-derived acidic region to promote ZYG-1 targeting and new centriole assembly. Analysis of a different crystal form of the Drosophila Plk4 (DmPlk4) CPB suggests that it also forms a Z-shaped dimer. Comparison of the ZYG-1 and DmPlk4 CPBs revealed structural changes in the ZYG-1 CPB that confer selectivity for binding SPD-2 over Asterless-derived acidic regions. Overall, our findings suggest a conserved mechanism for centriolar docking of Plk4 homologs that initiate daughter centriole assembly.

Although loss of CP110 is tolerated in Drosophila, CP110 is important for limiting centriole length, limiting centriolar microtubule length, and suppressing centriole overduplication when duplication proteins are overexpressed., CP110 is a conserved centriole protein implicated in the regulation of cell division, centriole duplication, and centriole length and in the suppression of ciliogenesis. Surprisingly, we report that mutant flies lacking CP110 (CP110Δ) were viable and fertile and had no obvious defects in cell division, centriole duplication, or cilia formation. We show that CP110 has at least three functions in flies. First, it subtly influences centriole length by counteracting the centriole-elongating activity of several centriole duplication proteins. Specifically, we report that centrioles are ∼10% longer than normal in CP110Δ mutants and ∼20% shorter when CP110 is overexpressed. Second, CP110 ensures that the centriolar microtubules do not extend beyond the distal end of the centriole, as some centriolar microtubules can be more than 50 times longer than the centriole in the absence of CP110. Finally, and unexpectedly, CP110 suppresses centriole overduplication induced by the overexpression of centriole duplication proteins. These studies identify novel and surprising functions for CP110 in vivo in flies.

SummaryPolo-like kinase 4 (Plk4) is a conserved master regulator of centriole assembly [1]. Previously, we found that Drosophila Plk4 protein levels are actively suppressed during interphase [2]. Degradation of interphase Plk4 prevents centriole overduplication and is mediated by the ubiquitin-ligase complex SCFSlimb/βTrCP [3, 4]. Since Plk4 stability depends on its activity [5, 6], we studied the consequences of inactivating Plk4 or perturbing its phosphorylation state within its Slimb-recognition motif (SRM). Mass spectrometry of in-vitro-phosphorylated Plk4 and Plk4 purified from cells reveals that it is directly responsible for extensively autophosphorylating and generating its Slimb-binding phosphodegron. Phosphorylatable residues within this regulatory region were systematically mutated to determine their impact on Plk4 protein levels and centriole duplication when expressed in S2 cells. Notably, autophosphorylation of a single residue (Ser293) within the SRM is critical for Slimb binding and ubiquitination. Our data also demonstrate that autophosphorylation of numerous residues flanking S293 collectively contribute to establishing a high-affinity binding site for SCFSlimb. Taken together, our findings suggest that Plk4 directly generates its own phosphodegron and can do so without the assistance of an additional kinase(s).

Summary Background Centrioles are cylindrical microtubule-based structures whose assembly is critical for the formation of cilia, flagella, and centrosomes. The centriole proximal region harbors a cartwheel that dictates the 9-fold symmetry of centrioles. Although the cartwheel architecture has been recently analyzed, how it connects to the peripheral microtubules is not understood. More generally, a high-resolution view of the proximal region of the centriole is lacking, thus limiting understanding of the underlying assembly mechanisms. Results We report the complete architecture of the Trichonympha centriole proximal region using cryotomography. The resulting 3D map reveals several features, including additional densities in the cartwheel that exhibit a 9-fold symmetrical arrangement, as well as the structure of the Pinhead and the A-C linker that connect to microtubules. Moreover, we uncover striking chiral features that might impart directionality to the entire centriole. Furthermore, we identify Trichonympha SAS-6 and demonstrate that it localizes to the cartwheel in vivo. Conclusions Our work provides unprecedented insight into the architecture of the centriole proximal region, which is key for a thorough understanding of the mechanisms governing centriole assembly.

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.