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1.
PLoS Biol ; 18(8): e3000762, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760088

RESUMO

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.


Assuntos
Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mitose , Proteínas Serina-Treonina Quinases/genética , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interfase , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Optogenética/métodos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
2.
Mol Cell ; 79(2): 342-358.e12, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645368

RESUMO

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.


Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
3.
Gene ; 760: 144989, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32717307

RESUMO

Kinesin 14 family member KIFC1 is a mitotic kinesin which contains a C-terminal motor domain and plays a vital role for clustering the amplified centrosomes. Overexpression of KIFC1 in prostate cancer (PCa) cells showed resistance to docetaxel (DTX). The present study revealed that small KIFC1 inhibitor AZ82 suppresed the transcription and translation of KIFC1 significantly in PCa cells. AZ82 inhibited the KIFC1 expression both in the cytoplasm and nucleus of PCa cells. Inhibition of KIFC1 by AZ82 caused multipolar mitosis in PCa cells via de-clustering the amplified centrosomes and decreased the rate of cancer cell growth and proliferation. Moreover, depletion of KIFC1 reduced cells entering the cell cycle and caused PCa cells death through apoptosis by increasing the expression of Bax and Cytochrome C. Thereby, KIFC1 silencing and inhibition decreased the PCa cells survival by inducing multipolar mitosis as well as apoptosis, suggesting inhibition of KIFC1 using AZ82 might be a strategy to treat PCa by controlling the cancer cell proliferation.


Assuntos
Alanina/análogos & derivados , Centrossomo/efeitos dos fármacos , Cinesina/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Piridinas/farmacologia , Alanina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/metabolismo , Dineínas/metabolismo , Humanos , Cinesina/genética , Cinesina/metabolismo , Masculino , Mitose/efeitos dos fármacos , Miosinas/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia
4.
Cancer Invest ; 38(5): 300-309, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32378982

RESUMO

Centrosome amplification leads to aberrant mitosis, giving rise to aneuploidy and it has been associated with poor prognosis in human cancers. This study aimed to evaluate the relationship between polyploidy, centrosome abnormalities, and response to endocrine treatment in progestin-induced mouse mammary carcinomas. We found cells with three or more centrosomes in the polyploid tumors. The endocrine unresponsive tumors showed a higher average number of centrosomes per cell than the responsive tumors. The results suggest an association between polyploidy and centrosome amplification with the resistance to endocrine therapy in this luminal breast cancer model.


Assuntos
Neoplasias da Mama/patologia , Carcinoma/patologia , Centrossomo/patologia , Hormônios/metabolismo , Aneuploidia , Animais , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Feminino , Amplificação de Genes/fisiologia , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Mitose/fisiologia , Poliploidia
5.
Nature ; 580(7801): 106-112, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238932

RESUMO

Radial glial progenitor cells (RGPs) are the major neural progenitor cells that generate neurons and glia in the developing mammalian cerebral cortex1-4. In RGPs, the centrosome is positioned away from the nucleus at the apical surface of the ventricular zone of the cerebral cortex5-8. However, the molecular basis and precise function of this distinctive subcellular organization of the centrosome are largely unknown. Here we show in mice that anchoring of the centrosome to the apical membrane controls the mechanical properties of cortical RGPs, and consequently their mitotic behaviour and the size and formation of the cortex. The mother centriole in RGPs develops distal appendages that anchor it to the apical membrane. Selective removal of centrosomal protein 83 (CEP83) eliminates these distal appendages and disrupts the anchorage of the centrosome to the apical membrane, resulting in the disorganization of microtubules and stretching and stiffening of the apical membrane. The elimination of CEP83 also activates the mechanically sensitive yes-associated protein (YAP) and promotes the excessive proliferation of RGPs, together with a subsequent overproduction of intermediate progenitor cells, which leads to the formation of an enlarged cortex with abnormal folding. Simultaneous elimination of YAP suppresses the cortical enlargement and folding that is induced by the removal of CEP83. Together, these results indicate a previously unknown role of the centrosome in regulating the mechanical features of neural progenitor cells and the size and configuration of the mammalian cerebral cortex.


Assuntos
Centrossomo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Células Ependimogliais/citologia , Células-Tronco Neurais/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Proliferação de Células , Centríolos/metabolismo , Córtex Cerebral/patologia , Feminino , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/patologia , Neurogênese
6.
PLoS Genet ; 16(3): e1008673, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203508

RESUMO

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.


Assuntos
Centrossomo/metabolismo , Eucromatina/genética , Heterocromatina/metabolismo , Animais , Estruturas Cromossômicas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/metabolismo , Genoma/genética , Heterocromatina/genética , Heterocromatina/ultraestrutura , Histonas/genética , Sequências Repetitivas de Ácido Nucleico/genética
7.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211895

RESUMO

NUP188 encodes a scaffold component of the nuclear pore complex (NPC) and has been implicated as a congenital heart disease gene through an ill-defined function at centrioles. Here, we explore the mechanisms that physically and functionally segregate Nup188 between the pericentriolar material (PCM) and NPCs. Pulse-chase fluorescent labeling indicates that Nup188 populates centrosomes with newly synthesized protein that does not exchange with NPCs even after mitotic NPC breakdown. In addition, the steady-state levels of Nup188 are controlled by the sensitivity of the PCM pool, but not the NPC pool, to proteasomal degradation. Proximity-labeling and super-resolution microscopy show that Nup188 is vicinal to the inner core of the interphase centrosome. Consistent with this, we demonstrate direct binding between Nup188 and Cep152. We further show that Nup188 functions in centriole duplication at or upstream of Sas6 loading. Together, our data establish Nup188 as a component of PCM needed to duplicate the centriole with implications for congenital heart disease mechanisms.


Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Mitose , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centríolos/genética , Células HeLa , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Ligação Proteica , Proteólise , Transdução de Sinais , Fatores de Tempo
8.
Nat Commun ; 11(1): 903, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060285

RESUMO

Centrosomes are essential organelles with functions in microtubule organization that duplicate once per cell cycle. The first step of centrosome duplication is the daughter centriole formation followed by the pericentriolar material recruitment to this centriole. This maturation step was termed centriole-to-centrosome conversion. It was proposed that CEP295-dependent recruitment of pericentriolar proteins drives centriole conversion. Here we show, based on the analysis of proteins that promote centriole biogenesis, that the developing centriole structure helps drive centriole conversion. Depletion of the luminal centriole protein CEP44 that binds to the A-microtubules and interacts with POC1B affecting centriole structure and centriole conversion, despite CEP295 binding to centrioles. Impairment of POC1B, TUBE1 or TUBD1, which disturbs integrity of centriole microtubules, also prevents centriole-to-centrosome conversion. We propose that the CEP295, CEP44, POC1B, TUBE1 and TUBD1 centriole biogenesis pathway that functions in the centriole lumen and on the cytoplasmic side is essential for the centriole-to-centrosome conversion.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Ciclo Celular/genética , Centríolos/genética , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Ligação Proteica , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
9.
Virology ; 541: 52-62, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32056715

RESUMO

Zika virus (ZIKV) nonstructural protein 5 (NS5) plays a critical role in viral RNA replication and mediates key virus-host cell interactions. As with other flavivirus NS5 proteins, ZIKV NS5 is primarily found in the nucleus. We previously reported that the NS5 protein of dengue virus, another flavivirus, localized to centrosomes during cell division. Here we show that ZIKV NS5 also relocalizes from the nucleus to centrosomes during mitosis. In infected cells with supernumerary centrosomes, NS5 was present at all centrosomes. Transient expression of NS5 in uninfected cells confirmed that centrosomal localization was independent of other viral proteins. Live-cell imaging demonstrated that NS5-GFP accumulated at centrosomes shortly after break down of nuclear membrane and remained there through mitosis. Cells expressing NS5-GFP took longer to complete mitosis than control cells. Finally, an analysis of ZIKV NS5 binding partners revealed several centrosomal proteins, providing potential direct links between NS5 and centrosomes.


Assuntos
Centrossomo/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células HEK293 , Humanos , Mitose
10.
Biol Cell ; 112(1): 22-37, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31789463

RESUMO

BACKGROUND: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes. RESULTS: We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein. CONCLUSIONS AND SIGNIFICANCE: Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species.


Assuntos
Proteínas de Transporte/metabolismo , Centríolos , Centrossomo , Tubulina (Proteína)/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Linhagem Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cílios/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Humanos , Microscopia Eletrônica , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , RNA Interferente Pequeno
11.
Am J Physiol Cell Physiol ; 318(1): C48-C62, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618077

RESUMO

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of ß-catenin. The treatment also activated ß-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or ß-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-ß-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of ß-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.


Assuntos
Centrossomo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteínas 14-3-3/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Glicemia/metabolismo , Centrossomo/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Exorribonucleases/metabolismo , Feminino , Receptores Frizzled/genética , Produtos Finais de Glicação Avançada/farmacologia , Células HCT116 , Humanos , Insulina/sangue , Camundongos Endogâmicos ICR , Ácido Palmítico/farmacologia , Ligação Proteica , Ratos , Proteínas Wnt/genética , Quinases Associadas a rho/metabolismo
12.
Oncogene ; 39(2): 399-413, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31477840

RESUMO

Localized, nonindolent prostate cancer (PCa) is characterized by large-scale genomic rearrangements, aneuploidy, chromothripsis, and other forms of chromosomal instability (CIN), yet how this occurs remains unclear. A well-established mechanism of CIN is the overproduction of centrosomes, which promotes tumorigenesis in various mouse models. Therefore, we developed a single-cell assay for quantifying centrosomes in human prostate tissue. Surprisingly, centrosome loss-which has not been described in human cancer-was associated with PCa progression. By chemically or genetically inducing centrosome loss in nontumorigenic prostate epithelial cells, mitotic errors ensued, producing aneuploid, and multinucleated cells. Strikingly, transient or chronic centrosome loss transformed prostate epithelial cells, which produced highly proliferative and poorly differentiated malignant tumors in mice. Our findings suggest that centrosome loss could create a cellular crisis with oncogenic potential in prostate epithelial cells.


Assuntos
Centrossomo/metabolismo , Instabilidade Genômica , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Aneuploidia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Fragmentação do DNA , Células Epiteliais/patologia , Humanos , Masculino , Camundongos , Mitose/genética , Próstata/patologia
13.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 72-83, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844893

RESUMO

Type 2 diabetes increases the risk for cancer. Centrosome amplification can initiate tumorigenesis. We have described that type 2 diabetes increases the centrosome amplification of peripheral blood mononuclear cells, with high glucose, insulin, and palmitic acid as the triggers, which suggests that centrosome amplification is a candidate biological mechanism linking diabetes to cancer. In this study, we aimed to further investigate the signaling pathways of the diabetes-associated centrosome amplification and to examine whether and how resveratrol inhibits the centrosome amplification. The results showed that treatment with high glucose, insulin, and palmitic acid, alone or in combination, could increase the protein levels of phospho-protein kinase C alpha (p-PKCα), phospho-p38 mitogen-activated protein kinases (p-p38), c-myc, and c-jun, as well as the mRNA levels of c-myc and c-jun. PKCα inhibitor could inhibit the treatment-induced increase in the protein levels of p-p38, c-myc, and c-jun. Inhibitor or siRNA of p38 was also able to inhibit the treatment-induced increase in the levels of p-p38, c-myc, and c-jun. Meanwhile, knockdown of c-myc or c-jun did not alter the treatment-induced increase in the phosphorylation of PKCα or p38. Importantly, inhibition of the phosphorylation of PKCα or p38 and knockdown of c-myc or c-jun could attenuate the centrosome amplification. In diabetic mice, the levels of p-PKCα, p-p38, c-myc, and c-jun were all increased in the colon tissues. Interestingly, resveratrol, but not metformin, was able to attenuate the treatment-induced increase in the levels of p-PKCα, p-p38, c-myc, and c-jun, as well as the centrosome amplification. In conclusion, our results suggest that PKCα-p38 to c-myc/c-jun is the signaling pathway of the diabetes-associated centrosome amplification, and resveratrol attenuates the centrosome amplification by inhibiting this signaling pathway.


Assuntos
Centrossomo/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Centrossomo/metabolismo , Colo/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Técnicas de Silenciamento de Genes , Glucose/farmacologia , Células HCT116 , Humanos , Insulina/farmacologia , Camundongos , Ácido Palmítico/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , Estreptozocina/efeitos adversos , Estreptozocina/farmacologia , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/genética
14.
Cancer Sci ; 111(2): 395-405, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31854049

RESUMO

The onset of breast cancer among young patients is a major issue in cancer etiology. Our previous study has shown that poor prognosis in young women with breast cancer is associated with lower expression of the microRNA miR-1285-5p. In this study, we showed that the expression of miR-1285-5p is lower in tumor tissues than in normal tissues. Accumulating evidence suggests that miR-1285-5p plays critical roles in various types of cancers. However, the functional role of miR-1285-5p in breast cancer remains to be elucidated. Here, we showed the tumor-suppressive role of miR-1285-5p and detailed its mechanism of action in breast cancer. Overexpression of miR-1285-5p significantly inhibited cell proliferation in breast cancer cells regardless of the tumor subtype. Among the target genes of miR-1285-5p, we found that transmembrane protein 194A (TMEM194A) was directly regulated by miR-1285-5p. Notably, separation of centrosomes from the nuclear envelope was observed upon knockdown of TMEM194A or overexpression of miR-1285-5p. In conclusion, our findings show that miR-1285-5p is a tumor suppressor via TMEM194A inhibition in breast cancer.


Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Proteínas Nucleares/genética , Proteína ran de Ligação ao GTP/genética , Regiões 3' não Traduzidas , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Centrossomo/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
15.
Elife ; 82019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31794381

RESUMO

Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Ocludina/metabolismo , Junções Íntimas/metabolismo , Aneuploidia , Animais , Apoptose , Sistemas CRISPR-Cas , Diferenciação Celular , Proliferação de Células , Centrossomo/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Edição de Genes , Humanos , Camundongos , Camundongos Knockout , Microcefalia/genética , Microcefalia/patologia , Microtúbulos/metabolismo , Mutagênese , Mutação , Neurogênese/genética , Neurogênese/fisiologia , Ocludina/genética , Fuso Acromático/metabolismo , Junções Íntimas/genética
16.
Sci Adv ; 5(8): eaav0318, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31803841

RESUMO

JLP belongs to the JIP family whose members serve as scaffolding proteins that link motor proteins and their cargo for intracellular transport. Although JLP is mainly cytoplasmic, it accumulates as a focus in the perinuclear region when stimulated by extracellular stimuli. Focus formation, which changes the nucleus shape and concentrates the nuclear pores, depends on p38MAPK activation and the dynein retrograde motor protein complex. Extracellular stimuli trigger the tethering of PLK1 to the centrosome by JLP, leading to centrosome maturation and microtubule array formation. The centrosome localization domain of JLP is important for the binding of the centrosome and the formation of the JLP focus and the microtubule array. Furthermore, the formation of the JLP focus and the microtubule array is interdependent and important for the transport of NF-κB p65 to the nucleus and its unloading therein. In conclusion, JLP exhibits multiple functions in the nuclear translocation of NF-κB p65.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Citoplasma/metabolismo , Microtúbulos/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Dineínas/metabolismo , Células HEK293 , Humanos , Cinesina/metabolismo , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Fator de Transcrição RelA
17.
BMC Mol Cell Biol ; 20(1): 58, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31823729

RESUMO

BACKGROUND: Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. RESULTS: Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. CONCLUSIONS: The results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.


Assuntos
Aurora Quinase A/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Motivos de Aminoácidos , Aurora Quinase A/genética , Centrossomo/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Fosforilação , Fuso Acromático/genética , Fuso Acromático/metabolismo , Tubulina (Proteína)/genética
18.
Int J Mol Sci ; 21(1)2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31861395

RESUMO

It has been four decades since the discovery of p53, the designated 'Guardian of the Genome'. P53 is primarily known as a master transcription factor and critical tumor suppressor, with countless studies detailing the mechanisms by which it regulates a host of gene targets and their consequent signaling pathways. However, transcription-independent functions of p53 also strongly define its tumor-suppressive capabilities and recent findings shed light on the molecular mechanisms hinted at by earlier efforts. This review highlights the transcription-independent mechanisms by which p53 influences the cellular response to genomic instability (in the form of replication stress, centrosome homeostasis, and transposition) and cell death. We also pinpoint areas for further investigation in order to better understand the context dependency of p53 transcription-independent functions and how these are perturbed when TP53 is mutated in human cancer.


Assuntos
Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Centrossomo/metabolismo , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Replicação do DNA , Regulação da Expressão Gênica , Recombinação Homóloga , Humanos , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Transcrição Genética , Proteína Supressora de Tumor p53/genética
19.
Int J Dev Biol ; 63(8-9-10): 509-519, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31840788

RESUMO

The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export. The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun1, as well as with the LEM/HeH-family protein Src1. Sun1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun1 usually forms a so-called LINC complex. Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in permeabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics.


Assuntos
Núcleo Celular/metabolismo , Dictyostelium/fisiologia , Membrana Nuclear/metabolismo , Centrômero/metabolismo , Centrossomo/metabolismo , Citoesqueleto/metabolismo , Dictyostelium/genética , Cinesina/metabolismo , Laminas/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Mitose , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Domínios Proteicos , Multimerização Proteica , Tubulina (Proteína)/química , Quinases da Família src/metabolismo
20.
Mol Cells ; 42(12): 840-849, 2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31722512

RESUMO

The spatiotemporal mitotic processes are controlled qualitatively by phosphorylation and qualitatively by ubiquitination. Although the SKP1-CUL1-F-box protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C) mainly mediate ubiquitin-dependent proteolysis of mitotic regulators, the E3 ligase for a large portion of mitotic proteins has yet to be identified. Here, we report c-Cbl as an E3 ligase that degrades DDA3, a protein involved in spindle dynamics. Depletion of c-Cbl led to increased DDA3 protein levels, resulting in increased recruitment of Kif2a to the mitotic spindle, a concomitant reduction in spindle formation, and chromosome alignment defects. Furthermore, c-Cbl depletion induced centrosome over-duplication and centriole amplification. Therefore, we concluded that c-Cbl controls spindle dynamics and centriole duplication through its E3 ligase activity against DDA3.


Assuntos
Centríolos/metabolismo , Mitose , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fuso Acromático/metabolismo , Ciclo Celular , Centrossomo/metabolismo , Células HeLa , Humanos , Cinesina/metabolismo , Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , RNA Interferente Pequeno , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
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