RESUMEN
Centrioles are found in the centrosome core and, as basal bodies, at the base of cilia and flagella. Centriole assembly and duplication is controlled by Polo-like-kinase 4 (Plk4): these processes fail if Plk4 is downregulated and are promoted by Plk4 overexpression. Here we show that the centriolar protein Asterless (Asl; human orthologue CEP152) provides a conserved molecular platform, the amino terminus of which interacts with the cryptic Polo box of Plk4 whereas the carboxy terminus interacts with the centriolar protein Sas-4 (CPAP in humans). Drosophila Asl and human CEP152 are required for the centrosomal loading of Plk4 in Drosophila and CPAP in human cells, respectively. Depletion of Asl or CEP152 caused failure of centrosome duplication; their overexpression led to de novo centriole formation in Drosophila eggs, duplication of free centrosomes in Drosophila embryos, and centrosome amplification in cultured Drosophila and human cells. Overexpression of a Plk4-binding-deficient mutant of Asl prevented centriole duplication in cultured cells and embryos. However, this mutant protein was able to promote microtubule organizing centre (MTOC) formation in both embryos and oocytes. Such MTOCs had pericentriolar material and the centriolar protein Sas-4, but no centrioles at their core. Formation of such acentriolar MTOCs could be phenocopied by overexpression of Sas-4 in oocytes or embryos. Our findings identify independent functions for Asl as a scaffold for Plk4 and Sas-4 that facilitates self-assembly and duplication of the centriole and organization of pericentriolar material.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular , Centrosoma/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Oocitos/citología , Oocitos/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Centrioles are essential for the formation of microtubule-derived structures, including cilia, flagella and centrosomes. These structures are involved in a variety of functions, from cell motility to division. In most dividing animal cells, centriole formation is coupled to the chromosome cycle. However, this is not the case in certain specialized divisions, such as meiosis, and in some differentiating cells. For example, oocytes loose their centrioles upon differentiation, whereas multiciliated epithelial cells make several of those structures after they exit the cell cycle. Aberrations of centriole number are seen in many cancer cells. Recent studies began to shed light on the molecular control of centriole number, its variations in development, and how centriole number changes in human disease. Here we review the recent developments in this field.
Asunto(s)
Centriolos/metabolismo , Centriolos/fisiología , Animales , Diferenciación Celular/genética , Movimiento Celular/genética , Centriolos/genética , Centrosoma/metabolismo , Cilios/genética , Cilios/metabolismo , Cilios/fisiología , Cricetinae , Células Epiteliales/metabolismo , Flagelos/genética , Flagelos/metabolismo , Humanos , Masculino , Meiosis , Microtúbulos/genética , Microtúbulos/metabolismoRESUMEN
Neuron morphology and function are highly dependent on proper organization of the cytoskeleton. In neurons, the centrosome is inactivated early in development, and acentrosomal microtubules are generated by mechanisms that are poorly understood. Here, we show that neuronal migration, development, and polarization depend on the multi-subunit protein HAUS/augmin complex, previously described to be required for mitotic spindle assembly in dividing cells. The HAUS complex is essential for neuronal microtubule organization by ensuring uniform microtubule polarity in axons and regulation of microtubule density in dendrites. Using live-cell imaging and high-resolution microscopy, we found that distinct HAUS clusters are distributed throughout neurons and colocalize with γ-TuRC, suggesting local microtubule nucleation events. We propose that the HAUS complex locally regulates microtubule nucleation events to control proper neuronal development.
Asunto(s)
Centrosoma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Animales , Axones/metabolismo , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Dendritas/metabolismo , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , EmbarazoRESUMEN
Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions.
Asunto(s)
Centriolos/genética , Proteínas de Drosophila/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Espermatogénesis/genética , Animales , Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Peroxisomas/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.
Asunto(s)
Axones/metabolismo , Proteínas del Citoesqueleto/metabolismo , Dendritas/metabolismo , Microtúbulos/metabolismo , Células Piramidales/metabolismo , Animales , Axones/ultraestructura , Dendritas/ultraestructura , Hipocampo/embriología , Hipocampo/metabolismo , Hipocampo/ultraestructura , Humanos , Proteínas Asociadas a Microtúbulos , Microtúbulos/ultraestructura , Células Piramidales/ultraestructura , RatasRESUMEN
Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/ßTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/ßTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. Here, we show that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/ßTrCP binding. We demonstrate a multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained and a sequence of events is observed before PLK4 can autodestruct. First, we show that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, we show the importance of PLK4-Slimb/ßTrCP regulation as it operates in both soma and germline. As ßTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.
Asunto(s)
Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Regulación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Serina/metabolismo , Treonina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Centrioles are essential for the formation of microtubule-derived structures, including cilia and centrosomes. Abnormalities in centrosome number and structure occur in many cancers and are associated with genomic instability. In most dividing animal cells, centriole formation is coordinated with DNA replication and is highly regulated such that only one daughter centriole forms close to each mother centriole. Centriole formation is triggered and dependent on a conserved kinase, SAK/PLK4. Downregulation and overexpression of SAK/PLK4 is associated with cancer in humans, mice, and flies. Here we show that centrosome amplification is normally inhibited by degradation of SAK/PK4 degradation, mediated by the SCF/Slimb ubiquitin ligase. This complex physically interacts with SAK/PLK4, and in its absence, SAK/PLK4 accumulates, leading to the striking formation of multiple daughter centrioles surrounding each mother. This interaction is mediated via a conserved Slimb binding motif in SAK/PLK4, mutations of which leads to centrosome amplification. This regulation is likely to be conserved, because knockout of the ortholog of Slimb, beta-Trcp1 in mice, also leads to centrosome amplification. Because the SCF/beta-Trcp complex plays an important role in cell-cycle progression, our results lead to new understanding of the control of centrosome number and how it may go awry in human disease.