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How diverse cell fates and complex forms emerge and feed back to each other to sculpt functional organs remains unclear. In the developing heart, the myocardium transitions from a simple epithelium to an intricate tissue that consists of distinct layers: the outer compact and inner trabecular layers. Defects in this process, which is known as cardiac trabeculation, cause cardiomyopathies and embryonic lethality, yet how tissue symmetry is broken to specify trabecular cardiomyocytes is unknown. Here we show that local tension heterogeneity drives organ-scale patterning and cell-fate decisions during cardiac trabeculation in zebrafish. Proliferation-induced cellular crowding at the tissue scale triggers tension heterogeneity among cardiomyocytes of the compact layer and drives those with higher contractility to delaminate and seed the trabecular layer. Experimentally, increasing crowding within the compact layer cardiomyocytes augments delamination, whereas decreasing it abrogates delamination. Using genetic mosaics in trabeculation-deficient zebrafish models-that is, in the absence of critical upstream signals such as Nrg-Erbb2 or blood flow-we find that inducing actomyosin contractility rescues cardiomyocyte delamination and is sufficient to drive cardiomyocyte fate specification, as assessed by Notch reporter expression in compact layer cardiomyocytes. Furthermore, Notch signalling perturbs the actomyosin machinery in cardiomyocytes to restrict excessive delamination, thereby preserving the architecture of the myocardial wall. Thus, tissue-scale forces converge on local cellular mechanics to generate complex forms and modulate cell-fate choices, and these multiscale regulatory interactions ensure robust self-organized organ patterning.
Assuntos
Retroalimentação Fisiológica , Coração/anatomia & histologia , Coração/embriologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Organogênese , Peixe-Zebra/embriologia , Actomiosina/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Modelos Animais , Receptores Notch/metabolismo , Transdução de Sinais , Peixe-Zebra/anatomia & histologiaRESUMO
In the 1990s, labs on both sides of the Atlantic performed the largest genetic mutagenesis screen at that time using an emerging model organism: the zebrafish. Led by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever in Boston, USA, these colossal screens culminated in 1996 with the publication of 37 articles in a special issue of Development, which remains the journal's largest issue to this day. To celebrate the anniversary of the zebrafish issue and reflect on the 25â years since its publication, five zebrafish researchers share what the issue means to them, how it has contributed to their career and its impact on the zebrafish community.
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Modelos Animais , Mutagênese/genética , Peixe-Zebra/genética , Animais , HumanosRESUMO
DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR) genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1 Gy (low) and 2.0 Gy (high) doses of γ-radiation and proliferated for 48 h and 72 h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM) method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1 Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72 h. At 2.0 Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48 h and 72 h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.
Assuntos
Dano ao DNA , Metilação de DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Raios gama/efeitos adversos , Leucócitos Mononucleares/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Adulto , Feminino , Humanos , MasculinoRESUMO
Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model.
Assuntos
Actomiosina/fisiologia , Junções Aderentes/fisiologia , Células Epiteliais/fisiologia , Mecanotransdução Celular/fisiologia , Contração Muscular/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Actomiosina/química , Junções Aderentes/química , Animais , Simulação por Computador , Células Epiteliais/química , Humanos , Modelos Biológicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Estresse Mecânico , Proteína rhoA de Ligação ao GTP/químicaRESUMO
BACKGROUND: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT). METHODS: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4 bp deletion in CDH1 exon 11. RESULTS: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed. CONCLUSIONS: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling.
Assuntos
Mama/metabolismo , Caderinas/genética , Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , Antígenos CD , Sequência de Bases , Mama/citologia , Caderinas/deficiência , Adesão Celular , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Humanos , Análise de Sequência de RNA , Deleção de SequênciaRESUMO
In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell-cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Caderinas/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Células MCF-7 , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Organ morphogenesis is multifaceted, multiscale, and fundamentally a robust process. Despite the complex and dynamic nature of embryonic development, organs are built with reproducible size, shape, and function, allowing them to support organismal growth and life. This striking reproducibility of tissue form exists because morphogenesis is not entirely hardwired. Instead, it is an emergent product of mechanochemical information flow, operating across spatial and temporal scales-from local cellular deformations to organ-scale form and function, and back. In this review, we address the mechanical basis of organ morphogenesis, as understood by observations and experiments in living embryos. To this end, we discuss how mechanical information controls the emergence of a highly conserved set of structural motifs that shape organ architectures across the animal kingdom: folds and loops, tubes and lumens, buds, branches, and networks. Moving forward, we advocate for a holistic conceptual framework for the study of organ morphogenesis, which rests on an interdisciplinary toolkit and brings the embryo center stage.
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Despite numerous advances in our understanding of zebrafish cardiac regeneration, an aspect that remains less studied is how regenerating cardiomyocytes invade, and eventually replace, the collagen-containing fibrotic tissue following injury. Here, we provide an in-depth analysis of the process of cardiomyocyte invasion using live-imaging and histological approaches. We observed close interactions between protruding cardiomyocytes and macrophages at the wound border zone, and macrophage-deficient irf8 mutant zebrafish exhibited defects in extracellular matrix (ECM) remodeling and cardiomyocyte protrusion into the injured area. Using a resident macrophage ablation model, we show that defects in ECM remodeling at the border zone and subsequent cardiomyocyte protrusion can be partly attributed to a population of resident macrophages. Single-cell RNA-sequencing analysis of cells at the wound border revealed a population of cardiomyocytes and macrophages with fibroblast-like gene expression signatures, including the expression of genes encoding ECM structural proteins and ECM-remodeling proteins. The expression of mmp14b , which encodes a membrane-anchored matrix metalloproteinase, was restricted to cells in the border zone, including cardiomyocytes, macrophages, fibroblasts, and endocardial/endothelial cells. Genetic deletion of mmp14b led to a decrease in 1) macrophage recruitment to the border zone, 2) collagen degradation at the border zone, and 3) subsequent cardiomyocyte invasion. Furthermore, cardiomyocyte-specific overexpression of mmp14b was sufficient to enhance cardiomyocyte invasion into the injured tissue and along the apical surface of the wound. Altogether, our data shed important insights into the process of cardiomyocyte invasion of the collagen-containing injured tissue during cardiac regeneration. They further suggest that cardiomyocytes and resident macrophages contribute to ECM remodeling at the border zone to promote cardiomyocyte replenishment of the fibrotic injured tissue.
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The epicardium, the outermost layer of the heart, is an important regulator of cardiac regeneration. However, a detailed understanding of the crosstalk between the epicardium and myocardium during development requires further investigation. Here, we generated three models of epicardial impairment in zebrafish by mutating the transcription factor genes tcf21 and wt1a, and ablating tcf21+ epicardial cells. Notably, all three epicardial impairment models exhibited smaller ventricles. We identified the initial cause of this phenotype as defective cardiomyocyte growth, resulting in reduced cell surface and volume. This failure of cardiomyocyte growth was followed by decreased proliferation and increased abluminal extrusion. By temporally manipulating its ablation, we show that the epicardium is required to support cardiomyocyte growth mainly during early cardiac morphogenesis. By transcriptomic profiling of sorted epicardial cells, we identified reduced expression of FGF and VEGF ligand genes in tcf21-/- hearts, and pharmacological inhibition of these signaling pathways in wild type partially recapitulated the ventricular growth defects. Taken together, these data reveal distinct roles of the epicardium during cardiac morphogenesis and signaling pathways underlying epicardial-myocardial crosstalk.
Assuntos
Miócitos Cardíacos , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Miócitos Cardíacos/metabolismo , Ligantes , Fator A de Crescimento do Endotélio Vascular/metabolismo , Pericárdio/metabolismo , Organogênese/genética , Coração/fisiologia , Miocárdio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Apicoplast, an essential organelle of human malaria parasite Plasmodium falciparum contains a â¼35 kb circular genome and is a possible target for therapy. Proteins required for the replication and maintenance of the apicoplast DNA are not clearly known. Here we report the presence of single-stranded DNA binding protein (SSB) in P falciparum. PfSSB is targeted to the apicoplast and it binds to apicoplast DNA. A strong ssDNA binding activity specific to SSB was also detected in P. falciparum lysate. Both the recombinant and endogenous proteins form tetramers and the homology modelling shows the presence of an oligosaccharide/oligonucleotide-binding fold responsible for ssDNA binding. Additionally, we used SSB as a tool to track the mechanism of delayed death phenomena shown by apicoplast targeted drugs ciprofloxacin and tetracycline. We find that the transport of PfSSB is severely affected during the second life cycle following drug treatment. Moreover, the translation of PfSSB protein and not the transcription of PfSSB seem to be down-regulated specifically during second life cycle although there is no considerable change in protein expression profile between drug-treated and untreated parasites. These results suggest dual control of translocation and translation of apicoplast targeted proteins behind the delayed death phenomena.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Antiprotozoários/farmacologia , Núcleo Celular/metabolismo , Ciprofloxacina/farmacologia , DNA de Protozoário/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Eritrócitos/parasitologia , Teste de Complementação Genética , Organelas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia Estrutural de ProteínaRESUMO
During cardiac development, endocardial cells (EdCs) produce growth factors to promote myocardial morphogenesis and growth. In particular, EdCs produce neuregulin which is required for ventricular cardiomyocytes (CMs) to seed the multicellular ridges known as trabeculae. Defects in neuregulin signaling, or in endocardial sprouting toward CMs, cause hypotrabeculation. However, the mechanisms underlying endocardial sprouting remain largely unknown. Here, we first show by live imaging in zebrafish embryos that EdCs interact with CMs via dynamic membrane protrusions. After touching CMs, these protrusions remain in close contact with their target despite the vigorous cardiac contractions. Loss of the CM-derived peptide Apelin, or of the Apelin receptor, which is expressed in EdCs, leads to reduced endocardial sprouting and hypotrabeculation. Mechanistically, neuregulin signaling requires endocardial protrusions to induce extracellular signal-regulated kinase (Erk) activity in CMs and trigger their delamination. Altogether, these data show that Apelin signaling-dependent endocardial protrusions modulate CM behavior during trabeculation.
Assuntos
Endocárdio , Peixe-Zebra , Animais , Apelina/metabolismo , Endocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Neurregulinas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The transformation of the heart from a simple tube to a complex organ requires the orchestration of several morphogenetic processes. Two structures critical for cardiac function, the cardiac valves and the trabecular network, are formed through extensive tissue morphogenesis-endocardial cell migration, deadhesion and differentiation into fibroblast-like cells during valve formation, and cardiomyocyte delamination and apico-basal depolarization during trabeculation. Here, we review current knowledge of how these specialized structures acquire their shape by focusing on the underlying cellular behaviors and molecular mechanisms, highlighting findings from in vivo models and briefly discussing the recent advances in cardiac cell culture and organoids.
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Miócitos Cardíacos , Organogênese , Movimento Celular , Valvas Cardíacas , MorfogêneseRESUMO
The transcription factor Snai1, a well-known regulator of epithelial-to-mesenchymal transition, has been implicated in early cardiac morphogenesis as well as in cardiac valve formation. However, a role for Snai1 in regulating other aspects of cardiac morphogenesis has not been reported. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required in cardiomyocytes for myocardial wall integrity. Loss of snai1b increases the frequency of cardiomyocyte extrusion away from the cardiac lumen. Extruding cardiomyocytes exhibit increased actomyosin contractility basally as revealed by enrichment of p-myosin and α-catenin epitope α-18, as well as disrupted intercellular junctions. Transcriptomic analysis of wild-type and snai1b mutant hearts revealed the dysregulation of intermediate filament genes, including desmin b (desmb) upregulation. Cardiomyocyte-specific desmb overexpression caused increased cardiomyocyte extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 maintains the integrity of the myocardial epithelium, at least in part by repressing desmb expression.
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Regulação da Expressão Gênica , Coração/fisiologia , Filamentos Intermediários/genética , Fatores de Transcrição da Família Snail/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Miocárdio/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
INTRODUCTION: Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. 177Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of 177Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells. MATERIALS AND METHODS: Nimotuzumab labeled with 177Lu was characterized by SE-HPLC. Specificity of 177Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of 177Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR. RESULTS: 177Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with 177Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of 177Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of 177Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of 177Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of 177Lu-Nimotuzumab dose. CONCLUSION: 177Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by 177Lu-Nimotuzumab with therapeutic gains.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Lutécio/uso terapêutico , Radioisótopos/uso terapêutico , Células A549 , Apoptose/efeitos da radiação , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta à Radiação , Receptores ErbB/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , HumanosRESUMO
RhoGTPase activation of non-muscle myosin II regulates cell division, extrusion, adhesion, migration, and tissue morphogenesis. However, the regulation of myosin II and mechanotransduction is not straightforward. Increasingly, the role of myosin II on the feedback regulation of RhoGTPase signaling is emerging. Indeed, myosin II controls RhoGTPase signaling through multiple mechanisms, namely contractility driven advection, scaffolding, and sequestration of signaling molecules. Here we discuss these mechanisms by which myosin II regulates RhoGTPase signaling in cell adhesion, migration, and tissue morphogenesis.
RESUMO
Rho kinases (ROCK1 and ROCK2) function downstream of the small GTPase RhoA to drive actomyosin cytoskeletal remodeling. It has often been believed that ROCK1 and ROCK2 may be functionally redundant, as they share a highly conserved kinase domain. However, in this study, we report differential functional effects for these ROCKs at the epithelial zonula adherens (ZA). Using specific siRNA, we found that ROCK1 depletion disrupted cadherin organization at the ZA, accompanied by loss of F-actin and NMIIA, whereas ROCK2 knockdown had no significant effect. Further, ROCK1, but not ROCK2, was necessary to stabilize GTP-RhoA at the ZA, thereby sustaining junctional tension and inhibiting intraepithelial cell movement. We also found that nonmuscle myosin IIA is a major determinant of ROCK1 cortical stability. Thus, despite sharing the catalytic domain with ROCK2, ROCK1 appears to be the dominant kinase essential for junctional integrity and contractile tension at epithelial ZA.
Assuntos
Junções Aderentes/metabolismo , Quinases Associadas a rho/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Junções Aderentes/enzimologia , Caderinas/metabolismo , Movimento Celular/fisiologia , Humanos , Células MCF-7 , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/fisiologia , Contração Muscular/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIB/metabolismo , Transdução de Sinais , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The ATP-dependent chromatin remodeling factors regulate gene expression. However, it is not known whether these factors regulate each other. Given the ability of these factors to regulate the accessibility of DNA to transcription factors, we postulate that one ATP-dependent chromatin remodeling factor should be able to regulate the transcription of another ATP-dependent chromatin remodeling factor. In this paper, we show that BRG1 and SMARCAL1, both members of the ATP-dependent chromatin remodeling protein family, regulate each other. BRG1 binds to the SMARCAL1 promoter, while SMARCAL1 binds to the brg1 promoter. During DNA damage, the occupancy of SMARCAL1 on the brg1 promoter increases coinciding with an increase in BRG1 occupancy on the SMARCAL1 promoter, leading to increased brg1 and SMARCAL1 transcripts respectively. This is the first report of two ATP-dependent chromatin remodeling factors regulating each other.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dano ao DNA , Regulação da Expressão Gênica , Células HeLa , Humanos , Regiões Promotoras Genéticas , Elementos Reguladores de TranscriçãoRESUMO
Non-muscle myosin II (NMII) motor proteins are responsible for generating contractile forces inside eukaryotic cells. There is also a growing interest in the capacity for these motor proteins to influence cell signaling through scaffolding, especially in the context of RhoA GTPase signaling. We previously showed that NMIIA accumulation and stability within specific regions of the cell cortex, such as the zonula adherens (ZA), allows the formation of a stable RhoA signaling zone. Now we demonstrate a key role for Coronin 1B in maintaining this junctional pool of NMIIA, as depletion of Coronin 1B significantly compromised myosin accumulation and stability at junctions. The loss of junctional NMIIA, upon Coronin 1B knockdown, perturbed RhoA signaling due to enhanced junctional recruitment of the RhoA antagonist, p190B Rho GAP. This effect was blocked by the expression of phosphomimetic MRLC-DD, thus reinforcing the central role of NMII in regulating RhoA signaling.
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4-Butirolactona/análogos & derivados , Junções Intercelulares/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , 4-Butirolactona/metabolismo , Actomiosina/metabolismo , Junções Aderentes/metabolismo , Células CACO-2 , Caderinas/metabolismo , Células Epiteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Células MCF-7 , Modelos Biológicos , Cadeias Leves de Miosina/metabolismo , Fenótipo , Estabilidade ProteicaRESUMO
We used a computational approach to analyze the biomechanics of epithelial cell aggregates-islands, stripes, or entire monolayers-that combines both vertex and contact-inhibition-of-locomotion models to include cell-cell and cell-substrate adhesion. Examination of the distribution of cell protrusions (adhesion to the substrate) in the model predicted high-order profiles of cell organization that agree with those previously seen experimentally. Cells acquired an asymmetric distribution of basal protrusions, traction forces, and apical aspect ratios that decreased when moving from the edge to the island center. Our in silico analysis also showed that tension on cell-cell junctions and apical stress is not homogeneous across the island. Instead, these parameters are higher at the island center and scale up with island size, which we confirmed experimentally using laser ablation assays and immunofluorescence. Without formally being a three-dimensional model, our approach has the minimal elements necessary to reproduce the distribution of cellular forces and mechanical cross-talk, as well as the distribution of principal stress in cells within epithelial cell aggregates. By making experimentally testable predictions, our approach can aid in mechanical analysis of epithelial tissues, especially when local changes in cell-cell and/or cell-substrate adhesion drive collective cell behavior.