RESUMEN
Centriole biogenesis, as in most organelle assemblies, involves the sequential recruitment of sub-structural elements that will support its function. To uncover this process, we correlated the spatial location of 24 centriolar proteins with structural features using expansion microscopy. A time-series reconstruction of protein distributions throughout human procentriole assembly unveiled the molecular architecture of the centriole biogenesis steps. We found that the process initiates with the formation of a naked cartwheel devoid of microtubules. Next, the bloom phase progresses with microtubule blade assembly, concomitantly with radial separation and rapid cartwheel growth. In the subsequent elongation phase, the tubulin backbone grows linearly with the recruitment of the A-C linker, followed by proteins of the inner scaffold (IS). By following six structural modules, we modeled 4D assembly of the human centriole. Collectively, this work provides a framework to investigate the spatial and temporal assembly of large macromolecules.
Asunto(s)
Centriolos , Microtúbulos , Centriolos/metabolismo , Humanos , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMEN
The accurate timing and execution of organelle biogenesis is crucial for cell physiology. Centriole biogenesis is regulated by Polo-like kinase 4 (Plk4) and initiates in S-phase when a daughter centriole grows from the side of a pre-existing mother. Here, we show that a Plk4 oscillation at the base of the growing centriole initiates and times centriole biogenesis to ensure that centrioles grow at the right time and to the right size. The Plk4 oscillation is normally entrained to the cell-cycle oscillator but can run autonomously of it-potentially explaining why centrioles can duplicate independently of cell-cycle progression. Mathematical modeling indicates that the Plk4 oscillation can be generated by a time-delayed negative feedback loop in which Plk4 inactivates the interaction with its centriolar receptor through multiple rounds of phosphorylation. We hypothesize that similar organelle-specific oscillations could regulate the timing and execution of organelle biogenesis more generally.
Asunto(s)
Relojes Biológicos/fisiología , Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Biogénesis de Organelos , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
Centriole duplication is tightly controlled to maintain correct centriole number through the cell cycle. Key to this is the regulated degradation of PLK4, the master regulator of centriole duplication. Here, we show that the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 localises to centrosomes during S-phase, where it is required for the maintenance of normal centriole number. Depletion of Tiam1 leads to an increase in centrosomal PLK4 and centriole overduplication, whereas overexpression of Tiam1 can restrict centriole overduplication. Ultimately, Tiam1 depletion leads to lagging chromosomes at anaphase and aneuploidy, which are potential drivers of malignant progression. The effects of Tiam1 depletion on centrosomal PLK4 levels and centriole overduplication can be rescued by re-expression of both wild-type Tiam1 and catalytically inactive (GEF*) Tiam1, but not by Tiam1 mutants unable to bind to the F-box protein ßTRCP (also known as F-box/WD repeat-containing protein 1A) implying that Tiam1 regulates PLK4 levels through promoting ßTRCP-mediated degradation independently of Rac1 activation.
Asunto(s)
Centriolos , Proteínas Serina-Treonina Quinasas , Ciclo Celular , Proteínas de Ciclo Celular/genética , CentrosomaRESUMEN
Breast cancer gene 1 (BRCA1) contributes to the regulation of centrosome number. We previously identified receptor for activated C kinase 1 (RACK1) as a BRCA1-interacting partner. RACK1, a scaffold protein that interacts with multiple proteins through its seven WD40 domains, directly binds to BRCA1 and localizes to centrosomes. RACK1 knockdown suppresses centriole duplication, whereas RACK1 overexpression causes centriole overduplication in a subset of mammary gland-derived cells. In this study, we showed that RACK1 binds directly to polo-like kinase 1 (PLK1) and Aurora A, and promotes the Aurora A-PLK1 interaction. RACK1 knockdown decreased phosphorylated PLK1 (p-PLK1) levels and the centrosomal localization of Aurora A and p-PLK1 in S phase, whereas RACK1 overexpression increased p-PLK1 level and the centrosomal localization of Aurora A and p-PLK1 in interphase, resulting in an increase of cells with abnormal centriole disengagement. Overexpression of cancer-derived RACK1 variants failed to enhance the Aurora A-PLK1 interaction, PLK1 phosphorylation and the centrosomal localization of p-PLK1. These results suggest that RACK1 functions as a scaffold protein that promotes the activation of PLK1 by Aurora A in order to promote centriole duplication.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Aurora Quinasa A/genética , Proteínas de Ciclo Celular/genética , Centriolos/genética , Centrosoma , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Quinasa Tipo Polo 1RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Ciclo Celular , Proteínas de Ciclo Celular/genética , Centriolos/genéticaRESUMEN
RNA-binding motif protein 10 (RBM10) primarily regulates alternative splicing of certain genes. Loss-of-function mutations in RBM10 have been frequently reported in patients with various cancers. However, how RBM10 levels affect cell proliferation and tumorigenesis remains unknown. To elucidate the role of RBM10 in cell proliferation, we established HepG2-RBM10 knockout cell lines and derivative doxycycline-inducible RBM10-expressing cells. RBM10 over-expression caused growth arrest in the M phase with a monopolar spindle because of impaired centriole duplication. Two RBM10 splicing mutants, one with F345A/F347A and the other with only the C-terminal half (401-930), were sufficient to cause growth arrest, whereas an RBM10 mutant with cytoplasmic localization forced by an NES did not show growth arrest. RBM10 over-expression induced the formation of many large nuclear domains containing RBM10, PLK4, STIL and SAS6, which are the regulatory proteins involved in centriole duplication. Consistently, the centrioles in the RBM10-over-expressing HepG2 cells lost PLK4 and STIL, accounting for the unsuccessful centriole duplication. In contrast, RBM10 depletion resulted in elevated levels of cytoplasmic PLK4 with a concomitant increase in the number of centrioles in HepG2 cells but not in A549 cells. Thus, nuclear RBM10 regulates normal chromosomal division in a cell-type-specific manner, independent of alternative RNA splicing.
Asunto(s)
Núcleo Celular/metabolismo , Centriolos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Carcinogénesis/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Células Hep G2 , Humanos , TranscriptomaRESUMEN
Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Prior work was substantially extended by showing CFI-400945 causes polyploidy, growth inhibition, and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with >4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in lung cancer cells. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was significantly inhibited at well-tolerated dosages. Phosphohistone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05). Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism cooperated with PLK4 inhibition. Taken together, PLK4 inhibition alone or as part of a combination regimen is a promising way to combat lung cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Indazoles/farmacología , Indoles/farmacología , Poliploidía , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Centrosoma , Regulación Neoplásica de la Expresión Génica , Humanos , Indazoles/uso terapéutico , Indoles/uso terapéutico , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismoRESUMEN
The centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4AS, which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas de Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Ciclo Celular/genética , Centriolos/genética , Proteínas del Citoesqueleto , Células HeLa , Humanos , Proteínas de Microtúbulos/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The centrosome is a small but important organelle that participates in centriole duplication, spindle formation, and ciliogenesis. Each event is regulated by key enzymatic reactions, but how these processes are integrated remains unknown. Recent studies have reported that ciliogenesis is controlled by distal appendage proteins such as FBF1, also known as Albatross. However, the precise role of Albatross in the centrosome cycle, including centriole duplication and centrosome separation, remains to be determined. Here, we report a novel function for Albatross at the proximal ends of centrioles. Using Albatross monospecific antibodies, full-length constructs, and siRNAs for rescue experiments, we found that Albatross mediates centriole duplication by recruiting HsSAS-6, a cartwheel protein of centrioles. Moreover, Albatross participates in centrosome separation during mitosis by recruiting Plk1 to residue S348 of Albatross after its phosphorylation. Taken together, our results show that Albatross is a novel protein that spatiotemporally integrates different aspects of centrosome function, namely ciliogenesis, centriole duplication, and centrosome separation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serina/metabolismo , Quinasa Tipo Polo 1RESUMEN
Centrioles are core components of centrosomes, the major microtubule-organizing centers of animal cells, and act as basal bodies for cilia formation. Control of centriole number is therefore crucial for genome stability and embryogenesis. Centriole duplication requires the serine/threonine protein kinase Plk4. Here, we identify Cep78 as a human centrosomal protein and a new interaction partner of Plk4. Cep78 is mainly a centriolar protein that localizes to the centriolar wall. Furthermore, we find that Plk4 binds to Cep78 through its N-terminal domain but that Cep78 is not an in vitro Plk4 substrate. Cep78 colocalizes with Plk4 at centrioles and is required for Plk4-induced centriole overduplication. Interestingly, upon depletion of Cep78, newly synthesized Plk4 is not localized to centrosomes. Our results suggest that the interaction between Cep78 and the N-terminal catalytic domain of Plk4 is a new and important element in the centrosome overduplication process.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células HeLa , Humanos , Interfase , Unión Proteica , Transporte de ProteínasRESUMEN
Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Centriolos/genética , Microtúbulos/genética , Animales , Proteínas Portadoras/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/ultraestructura , Centrosoma/metabolismo , Centrosoma/ultraestructura , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Unión Proteica/genética , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
For over a century, the abnormal movement or number of centrosomes has been linked with errors of chromosomes distribution in mitosis. While not essential for the formation of the mitotic spindle, the presence and location of centrosomes has a major influence on the manner in which microtubules interact with the kinetochores of replicated sister chromatids and the accuracy with which they migrate to resulting daughter cells. A complex network has evolved to ensure that cells contain the proper number of centrosomes and that their location is optimal for effective attachment of emanating spindle fibers with the kinetochores. The components of this network are regulated through a series of post-translational modifications, including ubiquitin and ubiquitin-like modifiers, which coordinate the timing and strength of signaling events key to the centrosome cycle. In this review, we examine the role of the ubiquitin system in the events relating to centriole duplication and centrosome separation, and discuss how the disruption of these functions impacts chromosome segregation.
Asunto(s)
Centrosoma/metabolismo , Segregación Cromosómica/fisiología , Cromosomas Humanos/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Animales , Cromosomas Humanos/genética , Humanos , Ubiquitina/genéticaRESUMEN
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.
Asunto(s)
Mariposas Diurnas/citología , Centriolos/metabolismo , Espermatogénesis , Animales , Mariposas Diurnas/genética , Mariposas Diurnas/metabolismo , Masculino , Mitosis , Espermatocitos/citología , Espermatocitos/metabolismoRESUMEN
Centriole biogenesis depends on the polo-like kinase (PLK4) and a small group of structural proteins. The spatiotemporal regulation of these proteins at pre-existing centrioles is essential to ensure that centriole duplication occurs once per cell cycle. Here, we report that phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C, hereafter referred to as PIPKIγ) plays an important role in centriole fidelity. PIPKIγ localized in a ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S and G2 phases, but not in M phase. This localization was dependent upon an association with centrosomal protein of 152 KDa (CEP152). Without detaining cells in S or M phase, the depletion of PIPKIγ led to centriole amplification in a manner that was dependent upon PLK4 and spindle assembly abnormal protein 6 homolog (SAS6). The expression of exogenous PIPKIγ reduced centriole amplification that occurred as a result of endogenous PIPKIγ depletion, hydroxyurea treatment or PLK4 overexpression, suggesting that PIPKIγ is likely to function at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic polo-box domain of PLK4 and that this binding reduced the kinase activity of PLK4. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication, which acts by modulating the homeostasis of PLK4 activity.
Asunto(s)
Centriolos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ubiquitinación , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , ProteolisisRESUMEN
Centrioles are microtubule-based core components of centrosomes and cilia. They are duplicated exactly once during S-phase progression. Central to formation of each new (daughter) centriole is the formation of a nine-fold symmetrical cartwheel structure onto which microtubule triplets are deposited. In recent years, a module comprising the protein kinase polo-like kinase 4 (PLK4) and the two proteins STIL and SAS-6 have been shown to stay at the core of centriole duplication. Depletion of any one of these three proteins blocks centriole duplication and, conversely, overexpression causes centriole amplification. In this short review article, we summarize recent insights into how PLK4, STIL and SAS-6 co-operate in space and time to form a new centriole. These advances begin to shed light on the very first steps of centriole biogenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Biológicos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Homología de Secuencia de AminoácidoRESUMEN
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, but the mechanism underlying its recruitment to mammalian centrioles is not understood. In flies, Plk4 recruitment depends on Asterless, whereas nematodes rely on a distinct protein, Spd-2. Here, we have explored the roles of two homologous mammalian proteins, Cep152 and Cep192, in the centriole recruitment of human Plk4. We demonstrate that Cep192 plays a key role in centrosome recruitment of both Cep152 and Plk4. Double-depletion of Cep192 and Cep152 completely abolishes Plk4 binding to centrioles as well as centriole duplication, indicating that the two proteins cooperate. Most importantly, we show that Cep192 binds Plk4 through an N-terminal extension that is specific to the largest isoform. The Plk4 binding regions of Cep192 and Cep152 (residues 190-240 and 1-46, respectively) are rich in negatively charged amino acids, suggesting that Plk4 localization to centrioles depends on electrostatic interactions with the positively charged polo-box domain. We conclude that cooperation between Cep192 and Cep152 is crucial for centriole recruitment of Plk4 and centriole duplication during the cell cycle.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Drosophila , Proteínas de Drosophila/genética , Células HEK293 , Células HeLa , Humanos , Nematodos , Unión Proteica/genética , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genéticaRESUMEN
In animal cells, the dysregulation of centrosome duplication and cohesion maintenance leads to abnormal spindle assembly and chromosomal instability, contributing to developmental disorders and tumorigenesis. However, the molecular mechanisms involved in maintaining accurate centrosome number control and tethering are not fully understood. Here, we identified coiled-coil domain-containing 102A (CCDC102A) as a centrosomal protein exhibiting a barrel-like structure in the proximal regions of parent centrioles, where it prevents centrosome overduplication by restricting interactions between Cep192 and Cep152 on centrosomes, thereby ensuring bipolar spindle formation. Additionally, CCDC102A regulates the centrosome linker by recruiting and binding C-Nap1; it is removed from the centrosome after Nek2A-mediated phosphorylation at the onset of mitosis. Overall, our results indicate that CCDC102A participates in controlling centrosome number and maintaining centrosome cohesion, suggesting that a well-tuned system regulates centrosome structure and function throughout the cell cycle.
Asunto(s)
Centrosoma , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Ciclo Celular , Centrosoma/metabolismo , Centriolos/metabolismo , Proteínas/metabolismoRESUMEN
Centrosomes nucleate and anchor microtubules and therefore play major roles in spindle formation and chromosome segregation during mitosis. Duplication of the centrosome occurs, similar to DNA, only once during the cell cycle. Aberration of the centrosome number is common in human tumors. At the core of centriole duplication is the conserved polo-like kinase 4, Plk4, and two structural proteins, STIL and Sas-6. In this review, I summarize and discuss developments in our understanding of the first steps of centriole duplication and their regulation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos , Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ciclo Celular/fisiología , Centriolos/metabolismo , Centrosoma/metabolismo , Humanos , Mitosis , Neoplasias/genética , Neoplasias/metabolismo , Quinasa Tipo Polo 1RESUMEN
Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new 'daughter' centriole is built orthogonally on one side of each radially symmetric 'mother' centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.
Asunto(s)
Centriolos , Proteínas de Drosophila , Animales , Centriolos/metabolismo , Centrosoma/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Membrana Nuclear/metabolismoRESUMEN
The centrosome is composed of a pair of centrioles and serves as the major microtubule-organizing center (MTOC) in cells. Centrosome dysfunction has been linked to autosomal recessive primary microcephaly (MCPH), which is a rare human neurodevelopmental disorder characterized by small brain size with intellectual disability. Recently, several mouse models carrying mutated genes encoding centrosomal proteins have been generated to address the genotype-phenotype relationships in MCPH. However, several human-specific features were not observed in the mouse models during brain development. Herein, we generated isogenic hiPSCs carrying the gene encoding centrosomal CPAP-E1235V mutant protein using the CRISPR-Cas9 genome editing system, and examined the phenotypic features of wild-type and mutant hiPSCs and their derived brain organoids. Our results showed that the CPAP-E1235V mutant perturbed the recruitment of several centriolar proteins involved in centriole elongation, including CEP120, CEP295, CENTROBIN, POC5, and POC1B, onto nascent centrioles, resulting in the production of short centrioles but long cilia. Importantly, our wild-type hiPSC-derived brain organoid recapitulated many cellular events seen in the developing human brain, including neuronal differentiation and cortical spatial lamination. Interestingly, hiPSC-CPAP-E1235V-derived brain organoids induced p53-dependent neuronal cell death, resulting in the production of smaller brain organoids that mimic the microcephaly phenotype. Furthermore, we observed that the CPAP-E1235V mutation altered the spindle orientation of neuronal progenitor cells and induced premature neuronal differentiation. In summary, we have shown that the hiPSC-derived brain organoid coupled with CRISPR/Cas9 gene editing technology can recapitulate the centrosome/centriole-associated MCPH pathological features. Possible mechanisms for MCPH with centriole/centrosome dysfunction are discussed.