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1.
Development ; 147(19)2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32928909

RESUMO

Precise patterning within the three-dimensional context of tissues, organs and embryos implies that cells can sense their relative position. During preimplantation development, outside and inside cells rely on apicobasal polarity and the Hippo pathway to choose their fate. Despite recent findings suggesting that mechanosensing might be central to this process, the relationship between blastomere geometry (i.e. shape and position) and the Hippo pathway effector YAP remains unknown. We used a highly quantitative approach to analyse information on the geometry and YAP localisation of individual blastomeres of mouse and human embryos. We identified the proportion of exposed cell surface area as most closely correlating with the nuclear localisation of YAP. To test this relationship, we developed several hydrogel-based approaches to alter blastomere geometry in cultured embryos. Unbiased clustering analyses of blastomeres from such embryos revealed that this relationship emerged during compaction. Our results therefore pinpoint the time during early embryogenesis when cells acquire the ability to sense changes in geometry and provide a new framework for how cells might integrate signals from different membrane domains to assess their relative position within the embryo.


Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/metabolismo , Animais , Blastômeros/citologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
2.
Proc Natl Acad Sci U S A ; 117(5): 2645-2655, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964836

RESUMO

The main risk factor for stomach cancer, the third most common cause of cancer death worldwide, is infection with Helicobacter pylori bacterial strains that inject cytotoxin-associated gene A (CagA). As the first described bacterial oncoprotein, CagA causes gastric epithelial cell transformation by promoting an epithelial-to-mesenchymal transition (EMT)-like phenotype that disrupts junctions and enhances motility and invasiveness of the infected cells. However, the mechanism by which CagA disrupts gastric epithelial cell polarity to achieve its oncogenicity is not fully understood. Here we found that the apoptosis-stimulating protein of p53 2 (ASPP2), a host tumor suppressor and an important CagA target, contributes to the survival of cagA-positive H. pylori in the lumen of infected gastric organoids. Mechanistically, the CagA-ASPP2 interaction is a key event that promotes remodeling of the partitioning-defective (PAR) polarity complex and leads to loss of cell polarity of infected cells. Blockade of cagA-positive H. pylori ASPP2 signaling by inhibitors of the EGFR (epidermal growth factor receptor) signaling pathway-identified by a high-content imaging screen-or by a CagA-binding ASPP2 peptide, prevents the loss of cell polarity and decreases the survival of H. pylori in infected organoids. These findings suggest that maintaining the host cell-polarity barrier would reduce the detrimental consequences of infection by pathogenic bacteria, such as H. pylori, that exploit the epithelial mucosal surface to colonize the host environment.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/metabolismo , Células Epiteliais/citologia , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Organoides/microbiologia , Antígenos de Bactérias/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas de Bactérias/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Humanos , Organoides/metabolismo , Ligação Proteica , Estômago/microbiologia
3.
Proc Natl Acad Sci U S A ; 115(41): 10375-10380, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30232257

RESUMO

Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.


Assuntos
Blastocisto/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Fertilização in vitro , Hidrogéis/química , Zona Pelúcida/fisiologia , Animais , Blastocisto/citologia , Células Cultivadas , Criopreservação , Transferência Embrionária , Embrião de Mamíferos/citologia , Feminino , Camundongos , Gravidez
4.
Semin Cell Dev Biol ; 47-48: 74-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26349030

RESUMO

The first lineage segregation event in mouse embryos produces two separate cell populations: inner cell mass and trophectoderm. This is understood to be brought about by cells sensing their position within the embryo and differentiating accordingly. The cellular and molecular underpinnings of this process remain under investigation and have variously been considered to be completely stochastic or alternately, subject to some predisposition set up at fertilisation or before. Here, we consider these views in light of recent publications, discuss the possible role of cell geometry and mechanical forces in this process and describe how modelling could contribute in addressing this issue.


Assuntos
Blastocisto/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Movimento Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Modelos Biológicos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
5.
Nat Struct Mol Biol ; 31(6): 964-976, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38789684

RESUMO

The mouse and human embryo gradually loses totipotency before diversifying into the inner cell mass (ICM, future organism) and trophectoderm (TE, future placenta). The transcription factors TFAP2C and TEAD4 with activated RHOA accelerate embryo polarization. Here we show that these factors also accelerate the loss of totipotency. TFAP2C and TEAD4 paradoxically promote and inhibit Hippo signaling before lineage diversification: they drive expression of multiple Hippo regulators while also promoting apical domain formation, which inactivates Hippo. Each factor activates TE specifiers in bipotent cells, while TFAP2C also activates specifiers of the ICM fate. Asymmetric segregation of the apical domain reconciles the opposing regulation of Hippo signaling into Hippo OFF and the TE fate, or Hippo ON and the ICM fate. We propose that the bistable switch established by TFAP2C and TEAD4 is exploited to trigger robust lineage diversification in the developing embryo.


Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição de Domínio TEA , Fator de Transcrição AP-2 , Fatores de Transcrição , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Humanos , Transdução de Sinais , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Via de Sinalização Hippo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Desenvolvimento Embrionário/genética
6.
J Biol Chem ; 286(50): 43039-44, 2011 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-21998301

RESUMO

Although kinase mutations have been identified in various human diseases, much less is known about protein phosphatases. Here, we show that all apoptosis-stimulating proteins of p53 (ASPP) family members can bind protein phosphatase 1 (PP1) via two distinct interacting motifs. ASPP2 interacts with PP1 through an RVXF PP1 binding motif, whereas the inhibitory member of the ASPP family (iASPP) interacts with PP1 via a noncanonical motif (RNYF) that is located within its Src homology 3 domain (SH3). Phe-815 is crucial in mediating iASPP/PP1 interaction, and iASPP(F815A) fails to inhibit the transcriptional and apoptotic function of p53. This study identifies iASPP as a new binding partner of PP1, interacting through a noncanonical PP1 binding motif.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligação Proteica , Proteína Fosfatase 1/genética , Proteínas Repressoras/genética
7.
Nat Commun ; 13(1): 3861, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35790717

RESUMO

Mammalian pre-implantation embryos accumulate substantial lipids, which are stored in lipid droplets (LDs). Despite the fundamental roles of lipids in many cellular functions, the significance of building-up LDs for the developing embryo remains unclear. Here we report that the accumulation and mobilization of LDs upon implantation are causal in the morphogenesis of the pluripotent epiblast and generation of the pro-amniotic cavity in mouse embryos, a critical step for all subsequent development. We show that the CIDEA protein, found abundantly in adipocytes, enhances lipid storage in blastocysts and pluripotent stem cells by promoting LD enlargement through fusion. The LD-stored lipids are mobilized into lysosomes at the onset of lumenogenesis, but without CIDEA are prematurely degraded by cytosolic lipases. Loss of lipid storage or inactivation of lipophagy leads to the aberrant formation of multiple cavities within disorganised epithelial structures. Thus, our study reveals an unexpected role for LDs in orchestrating tissue remodelling and uncovers underappreciated facets of lipid metabolism in peri-implantation development.


Assuntos
Gotículas Lipídicas , Metabolismo dos Lipídeos , Adipócitos/metabolismo , Animais , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos , Mamíferos , Camundongos , Morfogênese
8.
Nat Commun ; 13(1): 941, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177595

RESUMO

During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Epitélio/crescimento & desenvolvimento , Morfogênese , Proteínas Supressoras de Tumor/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células CACO-2 , Polaridade Celular , Cães , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Epitélio/metabolismo , Feminino , Gastrulação , Camadas Germinativas , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Transgênicos , Mutação , Linha Primitiva , Receptores de Neuropeptídeo Y/metabolismo , Estresse Mecânico , Junções Íntimas/metabolismo , Proteínas Supressoras de Tumor/genética
9.
Philos Trans R Soc Lond B Biol Sci ; 375(1809): 20190560, 2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-32829691

RESUMO

Advances in fluorescence microscopy approaches have made it relatively easy to generate multi-dimensional image volumes and have highlighted the need for flexible image analysis tools for the extraction of quantitative information from such data. Here we demonstrate that by focusing on simplified feature-based nuclear segmentation and probabilistic cytoplasmic detection we can create a tool that is able to extract geometry-based information from diverse mammalian tissue images. Our open-source image analysis platform, called 'SilentMark', can cope with three-dimensional noisy images and with crowded fields of cells to quantify signal intensity in different cellular compartments. Additionally, it provides tissue geometry related information, which allows one to quantify protein distribution with respect to marked regions of interest. The lightweight SilentMark algorithms have the advantage of not requiring multiple processors, graphics cards or training datasets and can be run even with just several hundred megabytes of memory. This makes it possible to use the method as a Web application, effectively eliminating setup hurdles and compatibility issues with operating systems. We test this platform on mouse pre-implantation embryos, embryonic stem cell-derived embryoid bodies and mouse embryonic heart, and relate protein localization to tissue geometry. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.


Assuntos
Embrião de Mamíferos/embriologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/métodos , Proteínas/análise , Animais , Núcleo Celular/fisiologia , Camundongos
10.
Dev Cell ; 50(2): 127-128, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31336094

RESUMO

In this issue of Developmental Cell, Hashimoto and Sasaki (2019) explore the role of the Hippo pathway in the establishment of naive pluripotency and cell competition in the epiblast. Their work gives insight into how the mouse embryo selects cells with the highest future potential, through the activity of YAP/TEAD.


Assuntos
Proteínas Serina-Treonina Quinases , Fatores de Transcrição , Animais , Camadas Germinativas , Camundongos , Transdução de Sinais
11.
Oncogene ; 23(56): 9007-16, 2004 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-15489900

RESUMO

ASPP1 and ASPP2 are both proteins that interact with p53 and enhance its ability to induce apoptosis by selectively elevating the expression of proapoptotic p53-responsive genes. iASPP(RAI) is a third member of the family that is the most conserved inhibitor of p53-mediated apoptosis. Here, we have described iASPP, a longer form of iASPP(RAI), which at 828 amino acids is more than twice the size of iASPP(RAI). Using two antibodies that recognize both iASPP and iASPP(RAI), we report that this longer form of iASPP is the predominant form of the molecule expressed in cells. Like iASPP(RAI), iASPP also binds to p53 and inhibits apoptosis induced by p53 overexpression. However, whereas iASPP(RAI) is predominantly nuclear, the N-terminus of iASPP is entirely cytoplasmic, and the longer iASPP is located in both the cytoplasm and the nucleus. The effect upon subcellular localization of the longer N-terminus of iASPP means that this new, longer form of the molecule may be subject to greater regulation and provides another layer in the control of p53-induced apoptosis.


Assuntos
Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Sequência de Bases , Proteínas de Transporte/química , Linhagem Celular Tumoral , Primers do DNA , Humanos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia
12.
PLoS One ; 9(10): e111384, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25360797

RESUMO

The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP in vitro, in epithelial cells, and in vivo, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Polaridade Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Células CACO-2 , Proteínas de Ciclo Celular , Humanos , Fosforilação , Proteína Fosfatase 1/metabolismo , Transporte Proteico , Junções Íntimas/metabolismo
13.
Nat Cell Biol ; 16(11): 1092-104, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25344754

RESUMO

Epithelial to mesenchymal transition (EMT), and the reverse mesenchymal to epithelial transition (MET), are known examples of epithelial plasticity that are important in kidney development and cancer metastasis. Here we identify ASPP2, a haploinsufficient tumour suppressor, p53 activator and PAR3 binding partner, as a molecular switch of MET and EMT. ASPP2 contributes to MET in mouse kidney in vivo. Mechanistically, ASPP2 induces MET through its PAR3-binding amino-terminus, independently of p53 binding. ASPP2 prevents ß-catenin from transactivating ZEB1, directly by forming an ASPP2-ß-catenin-E-cadherin ternary complex and indirectly by inhibiting ß-catenin's N-terminal phosphorylation to stabilize the ß-catenin-E-cadherin complex. ASPP2 limits the pro-invasive property of oncogenic RAS and inhibits tumour metastasis in vivo. Reduced ASPP2 expression results in EMT, and is associated with poor survival in hepatocellular carcinoma and breast cancer patients. Hence, ASPP2 is a key regulator of epithelial plasticity that connects cell polarity to the suppression of WNT signalling, EMT and tumour metastasis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Metástase Neoplásica , Fosforilação , Homeobox 1 de Ligação a E-box em Dedo de Zinco
14.
Dev Cell ; 19(1): 126-37, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20619750

RESUMO

Cell polarity plays a key role in the development of the central nervous system (CNS). Interestingly, disruption of cell polarity is seen in many cancers. ASPP2 is a haplo-insufficient tumor suppressor and an activator of the p53 family. In this study, we show that ASPP2 controls the polarity and proliferation of neural progenitors in vivo, leading to the formation of neuroblastic rosettes that resemble primitive neuroepithelial tumors. Consistent with its role in cell polarity, ASPP2 influences interkinetic nuclear migration and lamination during CNS development. Mechanistically, ASPP2 maintains the integrity of tight/adherens junctions. ASPP2 binds Par-3 and controls its apical/junctional localization without affecting its expression or Par-3/aPKC lambda binding. The junctional localization of ASPP2 and Par-3 is interdependent, suggesting that they are prime targets for each other. These results identify ASPP2 as a regulator of Par-3, which plays a key role in controlling cell proliferation, polarity, and tissue organization during CNS development.


Assuntos
Moléculas de Adesão Celular/fisiologia , Sistema Nervoso Central/embriologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Proteínas de Ciclo Celular , Polaridade Celular/fisiologia , Proliferação de Células , Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Primers do DNA/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/anormalidades , Neocórtex/embriologia , Neurônios/citologia , Neurônios/metabolismo , Gravidez , Ligação Proteica , Retina/anormalidades , Retina/embriologia , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
15.
EMBO J ; 24(15): 2768-82, 2005 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-16037820

RESUMO

We show here that the cell cycle-dependent DNA-binding and transcriptional activity of p53 correlates with E2F expression in human primary fibroblasts. E2F1 binds and stimulates DNA-binding, transactivation and apoptotic functions of p53 but not p63 and p73. E2F1 binds residues 347-370 of p53 and enhances nuclear retention of Ser315 phosphorylated p53. This regulation of p53 by E2F1 is cell cycle dependent, as the cellular distribution of Ser315 phosphorylated p53 is associated with the periodic expression of E2F and cyclin A throughout the cell cycle. This is the first demonstration that the activities of p53 are regulated during the cell cycle by E2F/p53 interactions and that phosphorylation of p53 at Ser315 is required for this regulation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Serina/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Genes Supressores de Tumor , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Transativadores/metabolismo , Transcrição Gênica , Proteína Tumoral p73 , Proteínas Supressoras de Tumor
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