Centriole Engagement 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.

Cep57 and Cep57L1 maintain centriole engagement in interphase to ensure centriole duplication cycle.

Centrioles duplicate in interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Codepletion of Cep57 and Cep57L1 induces precocious centriole disengagement in interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation.

Phosphorylation of keratin 18 serine 52 regulates mother-daughter centriole engagement and microtubule nucleation by cell cycle-dependent accumulation at the centriole.

Serine-52 (Ser52) is the major physiologic site of keratin 18 (K18) phosphorylation. Here, we report that serine-52 phosphorylated K18 (phospho-Ser52 K18) accumulated on centrosomes in a cell cycle-dependent manner. Moreover, we found that phospho-Ser52 K18 was locatedat theproximalendof themother centriole. Transfection with the K18 Ser52 → Ala (K18 S52A) mutant prevented centriole localization of phospho-Ser52 K18 and resulted in separation of the mother-daughter centrioles. Inhibition of microtubulepolymerization led to the disappearance of aggregated phospho-Ser52 K18 on the centrosome; removal of inhibitors resulted in reaccumulation of phospho-Ser52 K18 in microtubule-organizing centers. Transfection with a K18 S52A mutant inhibited microtubule nucleation. These results reveal a cell cycle-dependent change in centrosome localization of phospho-Ser52 k18 and strongly suggest that the phosphorylation status of Ser52 K18 of mother centrioles plays a critical role in maintaining a tight engagement between mother and daughter centrioles and also contributes to microtubule nucleation.

Piezo Mechanosensory Channels Control Centriole Engagement via Calcium Signaling at the Centrosome

Mechanotransduction is a process by which mechanical stimuli are converted into biochemical signals in cells to elicit different physiological functions, including embryogenesis, hearing, touch and muscle contractility. Piezo1 and 2 are evolutionarily conserved mechanosensory cation channels known to function on the cell surface by responding to external pressure and transducing a mechanically activated ion current. Piezo proteins have overlapping but distinct expression patterns in tissues and cells, and therefore exhibit both common and unique biological functions. Both gain- and loss-of-function PIEZO1and PIEZO2 mutations are associated with a number of severe human diseases.The majority of studies have focused on the Piezo mechanosensor function on the cell membrane, with transduction of MA currents to the cytoplasm in response to extracellular forces. Here, we show in myoblasts and multiple other cell types that Piezo proteins are also expressed intracellularly at the centrosome. Both Piezo loss-of-function and Piezo1 activation produce supernumerary centrosomes due to inappropriate centriole disengagement, leading to multi-polar spindles and a cell cycle defect. Some of the free centrioles migrate to the cell periphery to form supernumerary basal bodies and cilia. We further show that perturbations in Piezo modulate Calcium flux at centrosomes, indicated by a Calcium reporter, and that photoactivation of a caged Piezo1 agonist at centrosomes lead to rapid centriole disengagement. Thus, we suggest that mechanotransduction by Piezo in peri-centrosomal pool regulates centriole engagement by maintaining peri-centrosomal Calcium within a defined range, likely through sensing cell intrinsic forces from microtubules.

The Cep57-pericentrin module organizes PCM expansion and centriole engagement

Centriole duplication occurs once per cell cycle to ensure robust formation of bipolar spindles and chromosome segregation. Each newly-formed daughter centriole remains connected to its mother centriole until late mitosis. The disengagement of the centriole pair is required for centriole duplication. However, the mechanisms underlying centriole engagement remain poorly understood. Here, we show that Cep57 is required for pericentriolar material (PCM) organization that regulates centriole engagement. Depletion of Cep57 causes PCM disorganization and precocious centriole disengagement during mitosis. The disengaged daughter centrioles acquire ectopic microtubule-organizing-center activity, which results in chromosome mis-segregation. Similar defects are observed in mosaic variegated aneuploidy syndrome patient cells with cep57 mutations. We also find that Cep57 binds to the well-conserved PACT domain of pericentrin. Microcephaly osteodysplastic primordial dwarfism disease pericentrin mutations impair the Cep57-pericentrin interaction and lead to PCM disorganization. Together, our work demonstrates that Cep57 provides a critical interface between the centriole core and PCM.

Promotion and Suppression of Centriole Duplication Are Catalytically Coupled through PLK4 to Ensure Centriole Homeostasis

SUMMARYPLK4 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.

Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes.

Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing.

Degradation of Cep68 and PCNT cleavage mediate Cep215 removal from the PCM to allow centriole separation, disengagement and licensing.

An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(βTrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser332, allowing recognition by βTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215.We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.

Centriole Engagement: It’s Not Just Cohesin Any More

The belief that cohesin complexes link mother to daughter centrioles has received substantial experimental support. New studies challenge the primacy of cohesin in centriole engagement and provide a more nuanced view into the mechanisms for centriole disengagement in anaphase.

CDK5RAP2 Regulates Centriole Engagement and Cohesion in Mice

Centriole duplication occurs once per cell cycle, ensuring that each cell contains two centrosomes, each containing a mother-daughter pair of tightly engaged centrioles at mitotic entry. Loss of the tight engagement between mother and daughter centrioles appears to license the next round of centriole duplication. However, the molecular mechanisms regulating this process remain largely unknown. Mutations in CDK5RAP2, which encodes a centrosomal protein, cause autosomal recessive primary microcephaly in humans. Here we show that CDK5RAP2 loss of function in mice causes centriole amplification with a preponderance of single, unpaired centrioles and increased numbers of daughter-daughter centriole pairs. These results indicate that CDK5RAP2 is required to maintain centriole engagement and cohesion, thereby restricting centriole replication. Early in mitosis, amplified centrosomes assemble multipolar spindles in CDK5RAP2 mutant cells. Moreover, both mother and daughter centrioles are amplified and the excess mother centrioles template multiple primary cilia in CDK5RAP2 mutant cells.

Mechanism limiting centrosome duplication to once per cell cycle

The centrosome organizes the microtubule cytoskeleton and consists of a pair of centrioles surrounded by pericentriolar material. Cells begin the cell cycle with a single centrosome, which duplicates once before mitosis. During duplication, new centrioles grow orthogonally to existing ones and remain engaged (tightly opposed) with those centrioles until late mitosis or early G1 phase, when they become disengaged. The relationship between centriole engagement/disengagement and centriole duplication potential is not understood, and the mechanisms that control these processes are not known. Here we show that centriole disengagement requires the protease separase at anaphase, and that this disengagement licences centriole duplication in the next cell cycle. We describe an in vitro system using Xenopus egg extract and purified centrioles in which both centriole disengagement and centriole growth occur. Centriole disengagement at anaphase is independent of mitotic exit and Cdk2/cyclin E activity, but requires the anaphase-promoting complex and separase. In contrast to disengagement, new centriole growth occurs in interphase, is dependent on Cdk2/cyclin E, and requires previously disengaged centrioles. This suggests that re-duplication of centrioles within a cell cycle is prevented by centriole engagement itself. We propose that the 'once-only' control of centrosome duplication is achieved by temporally separating licensing in anaphase from growth of new centrioles during S phase. The involvement of separase in both centriole disengagement and sister chromatid separation would prevent premature centriole disengagement before anaphase onset, which can lead to multipolar spindles and genomic instability.