RESUMEN
In most animals, female meiotic spindles are assembled in the absence of centrosomes. How microtubules (MTs) are organized into acentrosomal meiotic spindles is poorly understood. In Caenorhabditis elegans, assembly of female meiotic spindles requires MEI-1 and MEI-2, which constitute the microtubule-severing AAA+ ATPase Katanin. However, the role of MEI-2 is not known and whether MT severing is required for meiotic spindle assembly is unclear. Here, we show that the essential role of MEI-2 is to confer MT binding to Katanin, which in turn stimulates the ATPase activity of MEI-1, leading to MT severing. To test directly the contribution of MT severing to meiotic spindle assembly, we engineered Katanin variants that retained MT binding and MT bundling activities but that were inactive for MT severing. In vivo analysis of these variants showed disorganized microtubules that lacked focused spindle poles reminiscent of the Katanin loss-of-function phenotype, demonstrating that the MT-severing activity is essential for meiotic spindle assembly in C. elegans Overall, our results reveal the essential role of MEI-2 and provide the first direct evidence supporting an essential role of MT severing in meiotic spindle assembly in C. elegans.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Adenosina Trifosfatasas/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Femenino , Katanina , Meiosis/genética , Meiosis/fisiología , Microtúbulos/genética , Huso Acromático/genéticaRESUMEN
The evolutionarily conserved microtubule (MT)-severing AAA-ATPase enzyme Katanin is emerging as a critical regulator of MT dynamics. In Caenorhabditis elegans, Katanin MT-severing activity is essential for meiotic spindle assembly but is toxic for the mitotic spindle. Here we analyzed Katanin dynamics in C. elegans and deciphered the role of Katanin phosphorylation in the regulation of its activity and stability. Katanin is abundant in oocytes, and its levels drop after meiosis, but unexpectedly, a significant fraction is present throughout embryogenesis, where it is dynamically recruited to the centrosomes and chromosomes during mitosis. We show that the minibrain kinase MBK-2, which is activated during meiosis, phosphorylates Katanin at multiple serines. We demonstrate unequivocally that Katanin phosphorylation at a single residue is necessary and sufficient to target Katanin for proteasomal degradation after meiosis, whereas phosphorylation at the other sites only inhibits Katanin ATPase activity stimulated by MTs. Our findings suggest that cycles of phosphorylation and dephosphorylation fine-tune Katanin level and activity to deliver the appropriate MT-severing activity during development.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Embrión no Mamífero/metabolismo , Katanina/metabolismo , Microtúbulos/metabolismo , Oocitos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Embrión no Mamífero/enzimología , Desarrollo Embrionario , Katanina/genética , Meiosis , Mitosis , Fosforilación , Proteínas Tirosina Quinasas/genética , Interferencia de ARNRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Mandibuloacral dysplasia syndromes are mainly due to recessive LMNA or ZMPSTE24 mutations, with cardinal nuclear morphological abnormalities and dysfunction. We report five homozygous null mutations in MTX2, encoding Metaxin-2 (MTX2), an outer mitochondrial membrane protein, in patients presenting with a severe laminopathy-like mandibuloacral dysplasia characterized by growth retardation, bone resorption, arterial calcification, renal glomerulosclerosis and severe hypertension. Loss of MTX2 in patients' primary fibroblasts leads to loss of Metaxin-1 (MTX1) and mitochondrial dysfunction, including network fragmentation and oxidative phosphorylation impairment. Furthermore, patients' fibroblasts are resistant to induced apoptosis, leading to increased cell senescence and mitophagy and reduced proliferation. Interestingly, secondary nuclear morphological defects are observed in both MTX2-mutant fibroblasts and mtx-2-depleted C. elegans. We thus report the identification of a severe premature aging syndrome revealing an unsuspected link between mitochondrial composition and function and nuclear morphology, establishing a pathophysiological link with premature aging laminopathies and likely explaining common clinical features.
Asunto(s)
Acroosteólisis/metabolismo , Predisposición Genética a la Enfermedad/genética , Lipodistrofia/metabolismo , Mandíbula/anomalías , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Acroosteólisis/diagnóstico por imagen , Acroosteólisis/genética , Acroosteólisis/patología , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/metabolismo , Animales , Apoptosis , Caenorhabditis elegans , Proliferación Celular , Niño , Regulación hacia Abajo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Genotipo , Homocigoto , Humanos , Lipodistrofia/diagnóstico por imagen , Lipodistrofia/genética , Lipodistrofia/patología , Masculino , Mandíbula/diagnóstico por imagen , Proteínas de la Membrana/genética , Metaloendopeptidasas , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas Mitocondriales/genética , Mutación , Fenotipo , Piel , Secuenciación Completa del GenomaRESUMEN
In animal cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Whereas mitotic kinases have been implicated in NEBD, how they coordinate their activity to trigger this event is unclear. Here, we show that both in human cells and Caenorhabditis elegans, the Polo-like kinase 1 (PLK-1) is recruited to the nuclear pore complexes, just prior to NEBD, through its Polo-box domain (PBD). We provide evidence that PLK-1 localization to the nuclear envelope (NE) is required for efficient NEBD. We identify the central channel nucleoporins NPP-1/Nup58, NPP-4/Nup54, and NPP-11/Nup62 as the critical factors anchoring PLK-1 to the NE in C. elegans. In particular, NPP-1, NPP-4, and NPP-11 primed at multiple Polo-docking sites by Cdk1 and PLK-1 itself physically interact with the PLK-1 PBD. We conclude that nucleoporins play an unanticipated regulatory role in NEBD, by recruiting PLK-1 to the NE thereby facilitating phosphorylation of critical downstream targets.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitosis/fisiología , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Células HeLa , Humanos , Membrana Nuclear/genética , Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Quinasa Tipo Polo 1RESUMEN
The conserved Bora protein is a Plk1 activator, essential for checkpoint recovery after DNA damage in human cells. Here, we show that Bora interacts with Cyclin B and is phosphorylated by Cyclin B/Cdk1 at several sites. The first 225 amino acids of Bora, which contain two Cyclin binding sites and three conserved phosphorylated residues, are sufficient to promote Plk1 phosphorylation by Aurora A in vitro. Mutating the Cyclin binding sites or the three conserved phosphorylation sites abrogates the ability of the N terminus of Bora to promote Plk1 activation. In human cells, Bora-carrying mutations of the three conserved phosphorylation sites cannot sustain mitotic entry after DNA damage. In C. elegans embryos, mutation of the three conserved phosphorylation sites in SPAT-1, the Bora ortholog, results in a severe mitotic entry delay. Our results reveal a crucial and conserved role of phosphorylation of the N terminus of Bora for Plk1 activation and mitotic entry.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/química , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/química , Secuencia Conservada , Ciclina B/metabolismo , Daño del ADN , Embrión no Mamífero/citología , Activación Enzimática , Células HeLa , Humanos , Mitosis , Fosforilación , Quinasa Tipo Polo 1RESUMEN
The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho-SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing.
Asunto(s)
Proteína Quinasa CDC2/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Aurora Quinasa A/metabolismo , Caenorhabditis elegans/enzimología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Activación Enzimática , Humanos , Larva/citología , Larva/enzimología , Mitosis , Datos de Secuencia Molecular , Fosforilación , Procesamiento Proteico-Postraduccional , Células Sf9 , SpodopteraRESUMEN
Molecular assays for monitoring sulfadoxine-pyrimethamine-resistant Plasmodium falciparum have not been implemented because of the genetic and statistical complexity of the parasite mutations that confer resistance and their relation to treatment outcomes. This study analyzed pretreatment dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes and treatment outcomes in a double-blind, placebo-controlled trial of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment for uncomplicated P. falciparum malaria. Multiple logistic regression was used to identify mutations that were predictive of treatment failure and to identify interactions and confounding factors. Infections caused by parasites with 3 DHFR mutations and 2 DHPS mutations (the "quintuple mutant") were associated with sulfadoxine-pyrimethamine treatment failure but not with chlorproguanil-dapsone treatment failure. The presence of a single DHFR mutation (Arg-59) with a single DHPS mutation (Glu-540) accurately predicted the presence of the quintuple mutant. If this model is validated in other populations, it will finally be possible to use molecular markers for surveillance of antifolate-resistant P. falciparum malaria in Africa.