Procentriole Formation Research Articles

Cartwheel disassembly regulated by CDK1-cyclin B kinase allows human centriole disengagement and licensing

Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis; however, the reason for persistence of the assembled human cartwheel structure from S phase to late mitosis remains unclear. Here, we demonstrate mainly using cell synchronization, RNA interference, immunofluorescence and time-lapse-microscopy, biochemical analysis, and methods that the cartwheel persistently assembles and maintains centriole engagement and centrosome integrity during S phase to late G2 phase. Blockade of the continuous accumulation of centriolar Sas-6, a major cartwheel protein, after procentriole formation induces premature centriole disengagement and disrupts pericentriolar matrix integrity. Additionally, we determined that during mitosis, CDK1-cyclin B phosphorylates Sas-6 at T495 and S510, disrupting its binding to cartwheel component STIL and pericentriolar component Nedd1 and promoting cartwheel disassembly and centriole disengagement. Perturbation of this phosphorylation maintains the accumulation of centriolar Sas-6 and retains centriole engagement during mitotic exit, which results in the inhibition of centriole reduplication. Collectively, these data demonstrate that persistent cartwheel assembly after procentriole formation maintains centriole engagement and that this configuration is relieved through phosphorylation of Sas-6 by CDK1-cyclin B during mitosis in human cells.

Interaction interface in the C-terminal parts of centriole proteins Sas6 and Ana2.

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.

Cep57 and Cep57l1 function redundantly to recruit the Cep63-Cep152 complex for centriole biogenesis.

The Cep63-Cep152 complex located at the mother centriole recruits Plk4 to initiate 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 loading of the Cep63-Cep152 complex to mother centrioles and consequently prevents centriole duplication. We propose that Cep57 and Cep57l1 function redundantly to ensure recruitment of the Cep63-Cep152 complex to the mother centrioles for procentriole formation.

First person – Daisuke Takao

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Daisuke Takao is first author on ‘Feedback loops in the Plk4–STIL–HsSAS6 network coordinate site selection for procentriole formation’, published in BiO. Daisuke conducted the research described in this article while an Assistant Professor in Daiju Kitagawa's lab at Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan. He is now at the Graduate School of Medicine, University of Tokyo, investigating cilia and centrosomes.

Self-organization of Plk4 regulates symmetry breaking in centriole duplication

During centriole duplication, a single daughter centriole is formed next to the mother centriole. The molecular mechanism that determines a single duplication site remains a long-standing question. Here, we show that intrinsic self-organization of Plk4 is implicated in symmetry breaking in the process of centriole duplication. We demonstrate that Plk4 has an ability to phase-separate into condensates via an intrinsically disordered linker and that the condensation properties of Plk4 are regulated by autophosphorylation. Consistently, the dissociation dynamics of centriolar Plk4 are controlled by autophosphorylation. We further found that autophosphorylated Plk4 is already distributed as a single focus around the mother centriole before the initiation of procentriole formation, and is subsequently targeted for STIL-HsSAS6 loading. Perturbation of Plk4 self-organization affects the asymmetry of centriolar Plk4 distribution and proper centriole duplication. Overall, we propose that the spatial pattern formation of Plk4 is a determinant of a single duplication site per mother centriole.

HsSAS-6-dependent cartwheel assembly ensures stabilization of centriole intermediates.

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.

Bimodal Binding of STIL to Plk4 Controls Proper Centriole Copy Number

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.

Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation

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.

Centrioles without microtubules: a new morphological type of centriole.

ABSTRACTThe 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.

Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation.

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.

NEK7 is required for G1 progression and procentriole formation.

The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin-CDK complexes. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The NIMA-related kinase NEK7 is one of many factors required for proper centriole duplication, as well as for timely cell cycle progression. However, its specific roles in these events are poorly understood. In this study, we find that depletion of NEK7 inhibits progression through the G1 phase in human U2OS cells via down-regulation of various cyclins and CDKs and also inhibits the earliest stages of procentriole formation. Depletion of NEK7 also induces formation of primary cilia in human RPE1 cells, suggesting that NEK7 acts at least before the restriction point during G1. G1-arrested cells in the absence of NEK7 exhibit abnormal accumulation of the APC/C cofactor Cdh1 at the vicinity of centrioles. Furthermore, the ubiquitin ligase APC/CCdh1 continuously degrades the centriolar protein STIL in these cells, thus inhibiting centriole assembly. Collectively our results demonstrate that NEK7 is involved in the timely regulation of G1 progression, S-phase entry, and procentriole formation.

Direct interaction of Plk4 with STIL ensures formation of a single procentriole per parental centriole.

Formation of one procentriole next to each pre-existing centriole is essential for centrosome duplication, robust bipolar spindle assembly and maintenance of genome integrity. However, the mechanisms maintaining strict control over centriole copy number are incompletely understood. Here we show that Plk4 and STIL, the key regulators of centriole formation, form a protein complex that provides a scaffold for recruiting HsSAS-6, a major component of the centriolar cartwheel, at the onset of procentriole formation. Furthermore, we demonstrate that phosphorylation of STIL by Plk4 facilitates the STIL/HsSAS-6 interaction and centriolar loading of HsSAS-6. We also provide evidence that negative feedback by centriolar STIL regulates bimodal centriolar distribution of Plk4 and seemingly restricts occurrence of procentriole formation to one site on each parental centriole. Overall, these findings suggest a mechanism whereby coordinated action of three critical factors ensures formation of a single procentriole per parental centriole.

Plk4 Phosphorylates Ana2 to Trigger Sas6 Recruitment and Procentriole Formation

SummaryCentrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer [1]. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms [2, 3, 4]. Although Plk4’s centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent.

Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells.

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.

Procentriole assembly without centriole disengagement - a paradox of male gametogenesis.

Disengagement of parent centrioles represents the licensing process to restrict centriole duplication exactly once during the cell cycle. However, we provide compelling evidence that this general rule is overridden in insect gametogenesis, when distinct procentrioles are generated during prophase of the first meiosis while parent centrioles are still engaged. Moreover, the number of procentrioles increases during the following meiotic divisions, and up to four procentrioles were found at the base of each mother centriole. However, procentrioles fail to organize a complete set of A-tubules and are thus unable to function as a template for centriole formation. Such a system, in which procentrioles form but halt growth, represents a unique model to analyze the process of cartwheel assembly and procentriole formation.

Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.

Tankyrase 1 regulates centrosome function by controlling CPAP stability

CPAP--a gene mutated in primary microcephaly--is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.

The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation

Centriole duplication involves the growth of a procentriole next to the parental centriole. Mutations in STIL and CPAP/CENPJ cause primary microcephaly (MCPH). Here, we show that human STIL has an asymmetric localization to the daughter centriole and is required for procentriole formation. STIL levels oscillate during the cell cycle. Interestingly, STIL interacts directly with CPAP and forms a complex with hSAS6. A natural mutation of CPAP (E1235V) that causes MCPH in humans leads to significantly lower binding to STIL. Overexpression of STIL induced the formation of multiple procentrioles around the parental centriole. STIL depletion inhibited normal centriole duplication, Plk4-induced centriole amplification, and CPAP-induced centriole elongation, and resulted in a failure to localize hSAS6 and CPAP to the base of the nascent procentriole. Furthermore, hSAS6 depletion hindered STIL targeting to the procentriole, implying that STIL and hSAS6 are mutually dependent for their centriolar localization. Together, our results indicate that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH.

PLK2 phosphorylation is critical for CPAP function in procentriole formation during the centrosome cycle

Control of centrosome duplication is tightly linked with the progression of the cell cycle. Recent studies suggest that the fundamental process of centriole duplication is evolutionally conserved. Here, we identified centrosomal P4.1-associated protein (CPAP), a human homologue of SAS-4, as a substrate of PLK2 whose activity oscillates during the cell cycle. PLK2 phosphorylates the S589 and S595 residues of CPAP in vitro and in vivo. This phosphorylation is critical for procentriole formation during the centrosome cycle. PLK4 also phosphorylates S595 of CPAP, but PLK4 phosphorylation is not a critical step in the PLK4 function in procentriole assembly. CPAP is phosphorylated in a cell cycle stage-specific manner, so that its phosphorylation increases at the G1/S transition phase and decreases during the exit of mitosis. Phosphorylated CPAP is preferentially located at the procentriole. Furthermore, overexpression of a phospho-resistant CPAP mutant resulted in the failure to form elongated centrioles. On the basis of these results, we propose that phosphorylated CPAP is involved in procentriole assembly, possibly for centriole elongation. This work demonstrates an example of how procentriole formation is linked to the progression of the cell cycle.

Procentriole assembly revealed by cryo-electron tomography

Centrosomes are cellular organelles that have a major role in the spatial organisation of the microtubule network. The centrosome is comprised of two centrioles that duplicate only once during the cell cycle, generating a procentriole from each mature centriole. Despite the essential roles of centrosomes, the detailed structural mechanisms involved in centriole duplication remain largely unknown. Here, we describe human procentriole assembly using cryo-electron tomography. In centrosomes, isolated from human lymphoblasts, we observed that each one of the nine microtubule triplets grows independently around a periodic central structure. The proximal end of the A-microtubule is capped by a conical structure and the B- and C-microtubules elongate bidirectionally from its wall. These observations suggest that the gamma tubulin ring complex (gamma-TuRC) has a fundamental role in procentriole formation by nucleating the A-microtubule that acts as a template for B-microtubule elongation that, in turn, supports C-microtubule growth. This study provides new insights into the initial structural events involved in procentriole assembly and establishes the basis for determining the molecular mechanisms of centriole duplication on the nanometric scale.