RESUMO
Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition.
Assuntos
Centrossomo/metabolismo , Replicação do DNA , Proteínas Repressoras/metabolismo , Animais , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Núcleo Celular/metabolismo , Cílios/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Vesículas Citoplasmáticas/metabolismo , Fibroblastos/metabolismo , Fase G1 , Células HEK293 , Células HeLa , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mitose , Mutação/genética , Fosforilação , Proteólise , Proteínas Repressoras/genética , Fase SRESUMO
BACKGROUND: Asthenoteratospermia, one of the most common causes for male infertility, often presents with defective sperm heads and/or flagella. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the common clinical manifestations of asthenoteratospermia. Variants in several genes including DNAH1, CEP135, CATSPER2 and SUN5 are involved in the genetic pathogenesis of asthenoteratospermia. However, more than half of the asthenoteratospermia cases cannot be explained by the known pathogenic genes. METHODS AND RESULTS: Two asthenoteratospermia-affected men with severe MMAF (absent flagella in >90% spermatozoa) from consanguineous families were subjected to whole-exome sequencing. The first proband had a homozygous missense mutation c.188G>A (p.Arg63Gln) of DZIP1 and the second proband had a homozygous stop-gain mutation c.690T>G (p.Tyr230*). Both of the mutations were neither detected in the human population genome data (1000 Genomes Project, Exome Aggregation Consortium) nor in our own data of a cohort of 875 Han Chinese control populations. DZIP1 encodes a DAZ (a protein deleted in azoospermia) interacting protein, which was associated with centrosomes in mammalian cells. Immunofluorescence staining of the centriolar protein Centrin1 indicated that the spermatozoa of the proband presented with abnormal centrosomes, including no concentrated centriolar dot or more than two centriolar dots. HEK293T cells transfected with two DZIP1-mutated constructs showed reduced DZIP1 level or truncated DZIP1. The Dzip1-knockout mice, generated by the CRSIPR-Cas9, revealed consistent phenotypes of severe MMAF. CONCLUSION: Our study strongly suggests that homozygous DZIP1 mutations can induce asthenoteratospermia with severe MMAF. The deficiency of DZIP1 induces sperm centrioles dysfunction and causes the absence of flagella.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Astenozoospermia/genética , Anormalidades Múltiplas/patologia , Animais , Astenozoospermia/patologia , Exoma/genética , Células HEK293 , Homozigoto , Humanos , Infertilidade Masculina , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Espermatozoides/metabolismo , Espermatozoides/patologia , Sequenciamento do ExomaRESUMO
The primary cilium, which disassembles before mitotic entry and reassembles after mitosis, organizes many signal transduction pathways that are crucial for cell life and individual development. However, how ciliogenesis is regulated during the cell cycle remains largely unknown. Here we show that GSK3ß, Dzip1, and Rab8 co-regulate ciliogenesis by promoting the assembly of the ciliary membrane after mitosis. Immunofluorescence and super-resolution microscopy showed that Dzip1 was localized to the periciliary diffusion barrier and enriched at the mother centriole. Knockdown of Dzip1 by short hairpin RNAs led to failed ciliary localization of Rab8, and Rab8 accumulation at the basal body. Dzip1 preferentially bound to Rab8GDP and promoted its dissociation from its inhibitor GDI2 at the pericentriolar region, as demonstrated by sucrose gradient centrifugation of purified basal bodies, immunoprecipitation, and acceptor-bleaching fluorescence resonance energy transfer assays. By means of in vitro phosphorylation, in vivo gel shift, phospho-peptide identification by mass spectrometry, and GST pulldown assays, we demonstrated that Dzip1 was phosphorylated by GSK3ß at S520 in G0 phase, which increased its binding to GDI2 to promote the release of Rab8GDP at the cilium base. Moreover, ciliogenesis was inhibited by overexpression of the GSK3ß-nonphosphorylatable Dzip1 mutant or by disabling of GSK3ß by specific inhibitors or knockout of GSK3ß in cells. Collectively, our data reveal a unique cascade consisting of GSK3ß, Dzip1, and Rab8 that regulates ciliogenesis after mitosis.
Assuntos
Cílios , Proteínas de Ligação a DNA/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Mitose , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Glicogênio Sintase Quinase 3 beta , Camundongos , Células NIH 3T3 , FosforilaçãoRESUMO
Centrosome duplication occurs under strict spatiotemporal regulation once per cell cycle, and it begins with cartwheel assembly and daughter centriole biogenesis at the lateral sites of the mother centrioles. However, although much of this process is understood, how centrosome duplication is initiated remains unclear. Here, we show that cartwheel assembly followed by daughter centriole biogenesis is initiated on the NEDD1-containing layer of the pericentriolar material (PCM) by the recruitment of SAS-6 to the mother centriole under the regulation of PLK4. We found that PLK4-mediated phosphorylation of NEDD1 at its S325 amino acid residue directly promotes both NEDD1 binding to SAS-6 and recruiting SAS-6 to the centrosome. Overexpression of phosphomimicking NEDD1 mutant S325E promoted cartwheel assembly and daughter centriole biogenesis initiations, whereas overexpression of nonphosphorylatable NEDD1 mutant S325A abolished the initiations. Collectively, our results demonstrate that PLK4-regulated NEDD1 facilitates initiation of the cartwheel assembly and of daughter centriole biogenesis in mammals.
Assuntos
Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismoRESUMO
A functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fuso Acromático/química , Fuso Acromático/metabolismo , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Dineínas/genética , Dineínas/metabolismo , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Fuso Acromático/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Centrosome number is tightly controlled during the cell cycle to ensure proper spindle assembly and cell division. However, the underlying mechanism that controls centrosome number remains largely unclear. We show herein that the DNA replication licensing factor Cdc6 is recruited to the proximal side of the centrioles via cyclin A to negatively regulate centrosome duplication by binding and inhibiting the cartwheel protein Sas-6 from forming a stable complex with another centriole duplication core protein, STIL. We further demonstrate that Cdc6 colocalizes with Plk4 at the centrosome, and interacts with Plk4 during S phase. Plk4 disrupts the interaction between Sas-6 and Cdc6, and suppresses the inhibitory role of Cdc6 on Sas-6 by phosphorylating Cdc6. Overexpressing wild-type Cdc6 or Plk4-unphosphorylatable Cdc6 mutant 2A reduces centrosome over-duplication caused by Plk4 overexpression or hydroxyurea treatment. Taken together, our data demonstrate that Cdc6 and Plk4 antagonistically control proper centrosome duplication during the cell cycle.