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1.
Viruses ; 12(6)2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32599823

RESUMO

The respiratory Influenza A Viruses (IAVs) and emerging zoonotic viruses such as Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pose a significant threat to human health. To accelerate our understanding of the host-pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (NHBE) cell culture models has gained prominence as an alternative to animal models. NHBE cells were differentiated under air-liquid interface (ALI) conditions to form an in vitro pseudostratified epithelium. The responses of well-differentiated (wd) NHBE cells were examined following infection with the 2009 pandemic Influenza A/H1N1pdm09 strain or following challenge with the dsRNA mimic, poly(I:C). At 30 h postinfection with H1N1pdm09, the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens-1 (ZO-1) from the cell cytoskeleton. wdNHBE cells produced an innate immune response to IAV-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding MUC5B, which may impair mucociliary clearance. Poly(I:C) produced similar responses to IAV, with the exception of MUC5B expression which was more than 3-fold higher than for control cells. This study demonstrates that wdNHBE cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Animais , Brônquios/citologia , Brônquios/virologia , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Cães , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/epidemiologia , Junções Intercelulares , Células Madin Darby de Rim Canino , Modelos Biológicos , Mucina-5AC/metabolismo , Pandemias , Cultura de Vírus
2.
PLoS Genet ; 16(6): e1008885, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559217

RESUMO

Regulation of cell junctions is crucial for the integrity of epithelial tissues and organs. Cell junctions also play roles in controlling cell proliferation for organ growth. Translationally controlled tumor protein (TCTP) is a conserved protein involved in growth control, but its role in cell junctions is unknown. Here we show that Drosophila Tctp directly interacts with the septate junction protein Coracle (Cora) to regulate epithelial integrity and organ growth. Tctp localizes together with Cora in the epidermis of the embryo. Loss of Cora reduces the level of Tctp in the epidermis but not vice versa. cora/+ or Tctp/+ single heterozygotes develop normally to adulthood. However, double heterozygotes for cora and Tctp mutations show severe disruption of epithelia causing synthetic lethality in the embryo. Double knockdown of Cora and Tctp in eye imaginal disc synergistically leads to disruption of the eye disc, resulting in a severe reduction or loss of eye and head. Conversely, double knockdown of Cora and Tctp in wing disc causes overgrowth as well as cell death. Inhibition of cell death under this condition causes hyperplastic growth of the wing disc. Tctp also shows direct and functional interaction with Cora-associated factors like Yurt and Na+/K+-ATPase. This study suggests that proper levels of Tctp and Cora are essential for the maintenance of the Cora complex and the integrity of epithelia. Our data also provide evidence that both Cora and Tctp are required to suppress overgrowth in developing wing.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Epitélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Proliferação de Células/genética , Proteínas de Drosophila/genética , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Discos Imaginais/crescimento & desenvolvimento , Junções Intercelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Morfogênese/genética , Mutações Sintéticas Letais , Asas de Animais/metabolismo
3.
Biochim Biophys Acta Biomembr ; 1862(6): 183237, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126234

RESUMO

Targeting the apical junctional complex during acute bacterial infections can be detrimental for the host in several aspects. First, the rupture of the epithelium or endothelium integrity is toxic in itself. In addition, extracellular bacterial pathogens or bacterial toxins can cross the body's physical barriers using the paracellular route and induce infection or intoxication of distant organs. No single strategy has been developed to disrupt junctional structures, rather each bacterium has its own method, which can be classed in one of the following three categories: (i) proteolysis/perturbation of adhesive proteins involved in tight or adherens junctions by bacterial or toxin-activated eukaryotic proteases, (ii) manipulation of host regulatory pathways leading to weakened intercellular adhesion, or (iii) delocalization of the junctional complex to open the gateway toward the subepithelial compartment. In this review, examples of each of these mechanisms are provided to illustrate how creative bacteria can be when seeking to disrupt cell-cell junctions.


Assuntos
Bactérias/patogenicidade , Junções Intercelulares/microbiologia , Animais , Infecções Bacterianas/etiologia , Toxinas Bacterianas/farmacologia , Humanos
4.
Am J Physiol Cell Physiol ; 318(6): C1046-C1054, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32130070

RESUMO

Cellular communication network (CCN) proteins are matricellular proteins that coordinate signaling among extracellular matrix, secreted proteins, and cell surface receptors. Their specific in vivo function is context-dependent, but they play profound roles in pathological conditions, such as fibrosis and cancers. Anti-CCN therapies are in clinical consideration. Only recently, however, has the function of these complex molecules begun to emerge. This review summarizes and interprets our current knowledge regarding these fascinating molecules and provides experimental evidence for their utility as therapeutic targets.


Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Comunicação Celular , Microambiente Celular , Matriz Extracelular/metabolismo , Junções Intercelulares/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Proteínas de Sinalização Intercelular CCN/genética , Matriz Extracelular/genética , Matriz Extracelular/patologia , Fibrose , Regulação Neoplásica da Expressão Gênica , Humanos , Junções Intercelulares/genética , Junções Intercelulares/patologia , Neoplasias/genética , Neoplasias/patologia , Microambiente Tumoral
5.
Biochim Biophys Acta Biomembr ; 1862(5): 183211, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32032590

RESUMO

Septate-like junctions display characteristic ladder-like ultrastructure reminiscent of the invertebrate epithelial septate junctions and are present at the paranodes of myelinated axons. The paranodal junctions where the myelin loops attach to the axon at the borders of the node of Ranvier provide both a paracellular barrier to ion diffusion and a lateral fence along the axonal membrane. The septate-like junctions constrain the proper distribution of nodal Na+ channels and juxtaparanodal K+ channels, which are required for the safe propagation of the nerve influx and rapid saltatory conduction. The paranodal cell adhesion molecules have been identified as target antigens in peripheral demyelinating autoimmune diseases and the pathogenic mechanisms described. This review aims at presenting the recent knowledge on the molecular and structural organization of septate-like junctions, their formation and stabilization during development, and how they are involved in demyelinating diseases.


Assuntos
Axônios/fisiologia , Fibras Nervosas Mielinizadas/metabolismo , Nós Neurofibrosos/metabolismo , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular/fisiologia , Humanos , Junções Intercelulares/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Nós Neurofibrosos/fisiologia , Vertebrados/metabolismo , Vertebrados/fisiologia
6.
J Immunol ; 204(4): 980-989, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31889022

RESUMO

Altered intestinal epithelial integrity is an important susceptibility trait in inflammatory bowel disease (IBD), and early life stressors are reported to contribute to this disease susceptibility in adulthood. To identify disease mechanisms associated with early-life trauma that exacerbate IBD in adulthood, we used a "double-hit" neonatal inflammation (NI) and adult inflammation (AI) model that exhibits more severe mucosal injury in the colon later in life. In this study, we explore the underlying mechanisms of this aggravated injury. In rats exposed to both NI and AI, we found sustained increases in colonic permeability accompanied by significantly attenuated expression of the epithelial junction protein E-cadherin. Quantitative RT-PCR revealed a decreased Cdh1 (gene of E-cadherin) mRNA expression in NI + AI rats compared with NI or AI rats. Next, we performed microRNA microarrays to identify potential regulators of E-cadherin in NI + AI rats. We confirmed the overexpression of miR-155, a predicted regulator of E-cadherin, and selected it for further analysis based on reported significance in human IBD. Using ingenuity pathway analysis software, the targets and related canonical pathway of miR-155 were analyzed. Mechanistic studies identified histone hyperacetylation at the Mir155 promoter in NI + AI rats, concomitant with elevated RNA polymerase II binding. In vitro, E-cadherin knockdown markedly increased epithelial cell permeability, as did overexpression of miR-155 mimics, which significantly suppressed E-cadherin protein. In vivo, NI + AI colonic permeability was significantly reversed with administration of miR-155 inhibitor rectally. Our collective findings indicate that early-life inflammatory stressors trigger a significant and sustained epithelial injury by suppressing E-cadherin through epigenetic mechanisms.


Assuntos
Caderinas/genética , Colo/imunologia , Epigênese Genética/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , MicroRNAs/metabolismo , Acetilação , Adulto , Animais , Caderinas/imunologia , Caderinas/metabolismo , Linhagem Celular , Colo/citologia , Colo/patologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Recém-Nascido , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Junções Intercelulares/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Masculino , MicroRNAs/antagonistas & inibidores , Permeabilidade/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ratos
7.
BMB Rep ; 53(2): 74-81, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31964473

RESUMO

Under physiological and pathological conditions, mechanical forces generated from cells themselves or transmitted from extracellular matrix (ECM) through focal adhesions (FAs) and adherens junctions (AJs) are known to play a significant role in regulating various cell behaviors. Substantial progresses have been made in the field of mechanobiology towards novel methods to understand how cells are able to sense and adapt to these mechanical forces over the years. To address these issues, this review will discuss recent advancements of traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM) to measure multiple aspects of cellular forces exerted by cells at cell-ECM and cell-cell junctional intracellular interfaces. We will also highlight how these methods can elucidate the roles of mechanical forces at interfaces of cell-cell/cell-ECM in regulating various cellular functions. [BMB Reports 2020; 53(2): 74-81].


Assuntos
Matriz Extracelular/fisiologia , Mecanotransdução Celular/fisiologia , Microscopia de Força Atômica/métodos , Biopolímeros , Adesão Celular/fisiologia , Matriz Extracelular/química , Adesões Focais/química , Adesões Focais/fisiologia , Hidrogéis , Junções Intercelulares/química , Junções Intercelulares/fisiologia , Estresse Mecânico , Tração
8.
Development ; 147(3)2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964776

RESUMO

Directional cell intercalations of epithelial cells during gastrulation has, in several organisms, been shown to be associated with a planar cell polarity in the organisation of the actin-myosin cytoskeleton and is postulated to reflect directional tension that drives oriented cell intercalations. We have characterised and applied a recently introduced non-destructive optical manipulation technique to measure the tension in individual epithelial cell junctions of cells in various locations and orientations in the epiblast of chick embryos in the early stages of primitive streak formation. Junctional tension of mesendoderm precursors in the epiblast is higher in junctions oriented in the direction of intercalation than in junctions oriented perpendicular to the direction of intercalation and higher than in junctions of other cells in the epiblast. The kinetic data fit best with a simple viscoelastic Maxwell model, and we find that junctional tension, and to a lesser extent viscoelastic relaxation time, are dependent on myosin activity.


Assuntos
Células Epiteliais/metabolismo , Gastrulação/fisiologia , Junções Intercelulares/metabolismo , Pinças Ópticas , Linha Primitiva/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Embrião de Galinha , Gástrula/metabolismo , Camadas Germinativas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrocarbonetos Clorados/farmacologia , Microscopia de Fluorescência/métodos , Miosina Tipo I/antagonistas & inibidores , Miosina Tipo I/metabolismo , Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/metabolismo , Pirróis/farmacologia , Transdução de Sinais/fisiologia
9.
Science ; 367(6477): 528-537, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31831638

RESUMO

Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia-neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury-induced changes at somatic junctions triggered P2Y12 receptor-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.


Assuntos
Lesões Encefálicas/imunologia , Encéfalo/imunologia , Junções Intercelulares/imunologia , Microglia/imunologia , Neurônios/imunologia , Receptores Purinérgicos P2Y12/fisiologia , Animais , Encéfalo/ultraestrutura , Lesões Encefálicas/patologia , Cálcio , Comunicação Celular/imunologia , Células HEK293 , Humanos , Camundongos , Mitocôndrias/imunologia , Canais de Potássio Shab/genética , Canais de Potássio Shab/fisiologia , Transdução de Sinais
10.
Toxicol In Vitro ; 62: 104682, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31626902

RESUMO

Roundup (R), a formulation that contains glyphosate (G) as the active ingredient, is a commonly used nonselective herbicide that has been proposed to affect male fertility. It is well known that an adequate Sertoli cell function is essential to maintain germ cell development. The aim of the present study was to analyze whether G and R are able to affect Sertoli cell functions, such as energy metabolism and blood-testis barrier (BTB) integrity. Sertoli cell cultures from 20-day-old rats were exposed to 10 and 100 ppm of G or R, doses which do not decrease cell viability. Neither G nor R caused impairment in lactate production or fatty acid oxidation. G and R decreased Transepithelial Electrical Resistance, which indicates the establishment of a Sertoli cell junction barrier. However, neither G nor R modified the expression of claudin11, ZO1 and occludin, proteins that constitute the BTB. Analysis of cellular distribution of claudin11 by immunofluorescence showed that G and R induced a delocalization of the signal from membrane to the cytoplasm. The results suggest that G and R could alter an important function of Sertoli cell such as BTB integrity and thus they could compromise the normal development of spermatogenesis.


Assuntos
Glicina/análogos & derivados , Herbicidas/toxicidade , Células de Sertoli/efeitos dos fármacos , Animais , Barreira Hematotesticular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Claudinas/biossíntese , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glicina/toxicidade , Junções Intercelulares/efeitos dos fármacos , Ácido Láctico/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Espermatogênese/efeitos dos fármacos
12.
Invest Ophthalmol Vis Sci ; 60(15): 5104-5111, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31826237

RESUMO

Purpose: Cell-cell contact in retinal pigment epithelium (RPE) involves adherent junctions, gap junctions, and tight junctions, which are primarily composed by E-cadherin, zona occludens 1 (ZO-1), and connexin 43, respectively. Here, we aimed to explore the relationship and interplay between these junction-associated proteins. Methods: E-cadherin, connexin 43, and ZO-1 expression in human primary RPE in the early phase after TGF-ß1 stimulation was detected. The knockdown of E-cadherin, ZO-1, and connexin 43 was performed to characterize the regulatory network involving these three proteins. Dye transfer and FITC-dextran permeability assays were conducted to observe the epithelial functional alterations. Transmission electron microscopy (TEM) was used to observe the ultrastructure of the cell-cell junctions in mouse RPE. The immunofluorescence staining and coimmunoprecipitation were performed to observe the colocalization and the physical association of E-cadherin, ZO-1, and connexin 43. Results: Among these three components, E-cadherin appeared to be the first protein that was downregulated after TGF-ß1 treatment. The ultrastructures of adherent junctions, gap junctions, and tight junctions could be observed in mouse RPE by TEM. E-cadherin, ZO-1, and connexin 43 were colocalized and physically bound to each other. The knockdown of one of these three proteins led to downregulation of the other two proteins and compromised epithelial function. Conclusions: E-cadherin, ZO-1, and connexin 43 were physically associated with each other and were mutually regulated. To enhance the understanding of cell-cell contacts, a holistic view is needed. Our results provide new insights in RPE disorders such as proliferative vitreoretinopathy.


Assuntos
Caderinas/genética , Conexina 43/genética , Regulação da Expressão Gênica , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/genética , Proteína da Zônula de Oclusão-1/genética , Animais , Caderinas/biossíntese , Células Cultivadas , Conexina 43/biossíntese , Humanos , Junções Intercelulares , Camundongos , Microscopia Eletrônica de Transmissão , RNA/genética , Epitélio Pigmentado da Retina/ultraestrutura , Junções Íntimas , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Proteína da Zônula de Oclusão-1/biossíntese
13.
PLoS Biol ; 17(12): e3000554, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790392

RESUMO

Junctional complexes between endothelial cells form a dynamic barrier that hinders passive diffusion of blood constituents into interstitial tissues. Remodelling of junctions is an essential process during leukocyte trafficking, vascular permeability, and angiogenesis. However, for many junctional proteins, the mechanisms of junctional remodelling have yet to be determined. Here, we used receptor mutagenesis, horseradish peroxidase (HRP), and ascorbate peroxidase 2 (APEX-2) proximity labelling, alongside light and electron microscopy (EM), to map the intracellular trafficking routes of junctional adhesion molecule-C (JAM-C). We found that JAM-C cotraffics with receptors associated with changes in permeability such as vascular endothelial cadherin (VE-Cadherin) and neuropilin (NRP)-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic JAM-C trafficking and degradation are necessary for junctional remodelling during cell migration and angiogenesis. By identifying new potential trafficking machinery, we show that a key point of regulation is the ubiquitylation of JAM-C by the E3 ligase Casitas B-lineage lymphoma (CBL), which controls the rate of trafficking versus lysosomal degradation.


Assuntos
Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Adesão Celular , Moléculas de Adesão Celular/fisiologia , Endotélio Vascular/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/fisiologia , Molécula C de Adesão Juncional , Leucócitos/fisiologia , Neuropilinas/metabolismo , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo
14.
Int J Mol Sci ; 21(1)2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31861297

RESUMO

The members of Rho family of GTPases, RhoA and Rac1 regulate endothelial cytoskeleton dynamics and hence barrier integrity. The spatial activities of these GTPases are regulated by post-translational prenylation. In the present study, we investigated the effect of prenylation inhibition on the endothelial cytoskeleton and barrier properties. The study was carried out in human umbilical vein endothelial cells (HUVEC) and protein prenylation is manipulated with various pharmacological inhibitors. Inhibition of either complete prenylation using statins or specifically geranylgeranylation but not farnesylation has a biphasic effect on HUVEC cytoskeleton and permeability. Short-term treatment inhibits the spatial activity of RhoA/Rho kinase (Rock) to actin cytoskeleton resulting in adherens junctions (AJ) stabilization and ameliorates thrombin-induced barrier disruption whereas long-term inhibition results in collapse of endothelial cytoskeleton leading to increased basal permeability. These effects are reversed by supplementing the cells with geranylgeranyl but not farnesyl pyrophosphate. Moreover, long-term inhibition of protein prenylation results in basal hyper activation of RhoA/Rock signaling that is antagonized by a specific Rock inhibitor or an activation of cAMP signaling. In conclusion, inhibition of geranylgeranylation in endothelial cells (ECs) exerts biphasic effect on endothelial barrier properties. Short-term inhibition stabilizes AJs and hence barrier function whereas long-term treatment results in disruption of barrier properties.


Assuntos
Endotélio/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Permeabilidade da Membrana Celular , Citoesqueleto/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Humanos , Junções Intercelulares/metabolismo , Modelos Biológicos , Prenilação de Proteína/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
15.
Phys Rev Lett ; 123(22): 228102, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31868410

RESUMO

Biological function requires cell-cell adhesions to tune their cohesiveness; for instance, during the opening of new fluid-filled cavities under hydraulic pressure. To understand the physical mechanisms supporting this adaptability, we develop a stochastic model for the hydraulic fracture of adhesive interfaces bridged by molecular bonds. We find that surface tension strongly enhances the stability of these interfaces by controlling flaw sensitivity, lifetime, and optimal architecture in terms of bond clustering. We also show that bond mobility embrittles adhesions and changes the mechanism of decohesion. Our study provides a mechanistic background to understand the biological regulation of cell-cell cohesion and fracture.


Assuntos
Adesão Celular/fisiologia , Junções Intercelulares/química , Junções Intercelulares/fisiologia , Modelos Biológicos , Membrana Celular/química , Membrana Celular/fisiologia , Simulação por Computador , Processos Estocásticos , Tensão Superficial
16.
Elife ; 82019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31697234

RESUMO

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting ßHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Membrana Celular/metabolismo , Polaridade Celular , Citoesqueleto/metabolismo , Drosophila melanogaster/ultraestrutura , Homeostase , Imageamento Tridimensional , Junções Intercelulares/metabolismo , Larva/citologia , Larva/ultraestrutura , Miosina Tipo V/metabolismo , Transporte Proteico , Glândulas Salivares/citologia , Glândulas Salivares/ultraestrutura , Proteínas rab de Ligação ao GTP/metabolismo
17.
Proc Natl Acad Sci U S A ; 116(48): 24108-24114, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31699818

RESUMO

Metastasis is the main cause of cancer-related deaths. How a single oncogenic cell evolves within highly organized epithelium is still unknown. Here, we found that the overexpression of the protein kinase atypical protein kinase C ι (aPKCi), an oncogene, triggers basally oriented epithelial cell extrusion in vivo as a potential mechanism for early breast tumor cell invasion. We found that cell segregation is the first step required for basal extrusion of luminal cells and identify aPKCi and vinculin as regulators of cell segregation. We propose that asymmetric vinculin levels at the junction between normal and aPKCi+ cells trigger an increase in tension at these cell junctions. Moreover, we show that aPKCi+ cells acquire promigratory features, including increased vinculin levels and vinculin dynamics at the cell-substratum contacts. Overall, this study shows that a balance between cell contractility and cell-cell adhesion is crucial for promoting basally oriented cell extrusion, a mechanism for early breast cancer cell invasion.


Assuntos
Neoplasias da Mama/metabolismo , Isoenzimas/fisiologia , Proteína Quinase C/fisiologia , Vinculina/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Separação Celular , Humanos , Junções Intercelulares/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Invasividade Neoplásica , Proteína Quinase C/genética , Proteína Quinase C/metabolismo
18.
Integr Biol (Camb) ; 11(1): 26-35, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31584068

RESUMO

The hypoxic microenvironment existing in vivo is known to significantly affect cell morphology and dynamics, and cell group behaviour. Collective migration of vascular endothelial cells is essential for vasculogenesis and angiogenesis, and for maintenance of monolayer integrity. Although hypoxic stress increases vascular endothelial permeability, the changes in collective migration and intracellular junction morphology of vascular endothelial cells remain poorly understood. This study reveals the migration of confluent vascular endothelial cells and changes in their adherens junction, as reflected by changes in the vascular endothelial (VE)-cadherin distribution, under hypoxic exposure. Vascular endothelial monolayers of human umbilical vein endothelial cells (HUVECs) were formed in microfluidic devices with controllability of oxygen tension. The oxygen tension was set to either normoxia (21% O2) or hypoxia (<3% O2) by supplying gas mixtures into separate gas channels. The migration velocity of HUVECs was measured using particle image velocimetry with a time series of phase-contrast microscopic images of the vascular endothelial monolayers. Hypoxia inducible factor-1α (HIF-1α) and VE-cadherin in HUVECs were observed after exposure to normoxic or hypoxic conditions using immunofluorescence staining and quantitative confocal image analysis. Changes in the migration speed of HUVECs were observed in as little as one hour after exposure to hypoxic condition, showing that the migration speed was increased 1.4-fold under hypoxia compared to that under normoxia. Nuclear translocation of HIF-1α peaked after the hypoxic gas mixture was supplied for 2 h. VE-cadherin expression was also found to be reduced. When ethanol was added to the cell culture medium, cell migration increased. By contrast, by strengthening VE-cadherin junctions with forskolin, cell migration decreased gradually in spite the effect of ethanol to stimulate migration. These results indicate that the increase of cell migration by hypoxic exposure was attributable to loosening of intercellular junction resulting from the decrease of VE-cadherin expression.


Assuntos
Junções Aderentes/metabolismo , Hipóxia Celular , Movimento Celular , Células Endoteliais da Veia Umbilical Humana/citologia , Transporte Ativo do Núcleo Celular , Antígenos CD/metabolismo , Caderinas/metabolismo , Colforsina/farmacologia , Endotélio Vascular/citologia , Etanol/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Processamento de Imagem Assistida por Computador , Junções Intercelulares/metabolismo , Dispositivos Lab-On-A-Chip , Microfluídica , Microscopia de Contraste de Fase , Oxigênio/metabolismo
19.
Nat Commun ; 10(1): 4113, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511517

RESUMO

Intra-organ communication guides morphogenetic processes that are essential for an organ to carry out complex physiological functions. In the heart, the growth of the myocardium is tightly coupled to that of the endocardium, a specialized endothelial tissue that lines its interior. Several molecular pathways have been implicated in the communication between these tissues including secreted factors, components of the extracellular matrix, or proteins involved in cell-cell communication. Yet, it is unknown how the growth of the endocardium is coordinated with that of the myocardium. Here, we show that an increased expansion of the myocardial atrial chamber volume generates higher junctional forces within endocardial cells. This leads to biomechanical signaling involving VE-cadherin, triggering nuclear localization of the Hippo pathway transcriptional regulator Yap1 and endocardial proliferation. Our work suggests that the growth of the endocardium results from myocardial chamber volume expansion and ends when the tension on the tissue is relaxed.


Assuntos
Endocárdio/crescimento & desenvolvimento , Miocárdio/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologia , Animais , Antígenos CD/metabolismo , Fenômenos Biomecânicos , Caderinas/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Tamanho Celular , Proteínas do Citoesqueleto/metabolismo , Endocárdio/citologia , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Junções Intercelulares/metabolismo , Modelos Biológicos , Mutação/genética , Transativadores/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo
20.
Physiol Int ; 106(3): 225-235, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31560236

RESUMO

OBJECTIVES: Impaired intestinal barrier function has been demonstrated in the pathophysiology of diarrhea-predominant irritable bowel syndrome (IBS-D). This study aimed to describe the intestinal ultrastructural findings in the intestinal mucosal layer of IBS-D patients. METHODS: In total, 10 healthy controls and 10 IBS-D patients were analyzed in this study. The mucosa of each patient's rectosigmoid colon was first assessed by confocal laser endomicroscopy (CLE); next, biopsied specimens of these sites were obtained. Intestinal tissues of IBS-D patients and healthy volunteers were examined to observe cellular changes by transmission electron microscopy (TEM). RESULTS: CLE showed no visible epithelial damage or inflammatory changes in the colonic mucosa of IBS-D compared with healthy volunteers. On transmission electron microscopic examination, patients with IBS-D displayed a larger apical intercellular distance with a higher proportion of dilated (>20 nm) intercellular junctional complexes, which was indicative of impaired mucosal integrity. In addition, microvillus exfoliation, extracellular vesicle as well as increased presence of multivesicular bodies were visible in IBS-D patients. Single epithelial cells appeared necrotic, as characterized by cytoplasmic vacuolization, cytoplasmic swelling, and presence of autolysosome. A significant association between bowel habit, frequency of abdominal pain, and enlarged intercellular distance was found. CONCLUSION: This study showed ultrastructural alterations in the architecture of intestinal epithelial cells and intercellular junctional complexes in IBS-D patients, potentially representing a pathophysiological mechanism in IBS-D.


Assuntos
Diarreia/patologia , Mucosa Intestinal/ultraestrutura , Síndrome do Intestino Irritável/patologia , Dor Abdominal/patologia , Colo Sigmoide/ultraestrutura , Citoplasma/patologia , Citoplasma/ultraestrutura , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Feminino , Humanos , Junções Intercelulares/ultraestrutura , Masculino , Pessoa de Meia-Idade , Reto/patologia , Reto/ultraestrutura
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