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1.
Cell ; 183(7): 1930-1945.e23, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33188777

RESUMO

RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.


Assuntos
Biossíntese de Proteínas , Vírus de RNA/fisiologia , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Interferons/metabolismo , Transporte de RNA , RNA Viral/genética , Reprodutibilidade dos Testes , Imagem Individual de Molécula , Fatores de Tempo
2.
Nat Microbiol ; 8(11): 2115-2129, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37814072

RESUMO

Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of this heterogeneity, we combined Virus Infection Real-time IMaging (VIRIM), a live-cell single-molecule imaging method, with real-time readouts of the dsRNA sensing pathway to analyse the response of human cells to encephalomyocarditis virus (EMCV) infection. We find that cell-to-cell heterogeneity in viral replication rates early in infection affect the efficiency of antiviral response activation, with lower replication rates leading to more antiviral response activation. Furthermore, we show that qualitatively distinct antiviral responses can be linked to the strength of the antiviral signalling pathway. Our analyses identify variation in early viral replication rates as an important parameter contributing to heterogeneity in antiviral response activation.


Assuntos
Viroses , Replicação Viral , Humanos , Transdução de Sinais , Vírus da Encefalomiocardite/fisiologia , Antivirais
3.
Free Radic Biol Med ; 206: 134-142, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37392950

RESUMO

Reactive Oxygen Species (ROS) in the form of H2O2 can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H2O2 were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2 production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H2O2 formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H2O2. However, a method to directly quantify the amount of H2O2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2 production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, to estimate whether the ensuing level of H2O2 production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTERT cells, addition of 5 mM d-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H2O2. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H2O2 production to be able to discriminate the effects of H2O2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.


Assuntos
Aminoácidos , Peróxido de Hidrogênio , Humanos , Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aminoácidos/metabolismo , Consumo de Oxigênio , Oxigênio
4.
PLoS One ; 13(11): e0207159, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30408122

RESUMO

Apico-basal polarity establishment is a seminal process in tissue morphogenesis. To function properly it is often imperative that epithelial cells limit apical membrane formation to a single domain. We previously demonstrated that signaling by the small GTPase Cdc42, together with its guanine nucleotide exchange factor (GEF) Tuba, is required to prevent the formation of multiple apical domains in polarized Ls174T:W4 cells, a single cell model for enterocyte polarization. To further chart the molecular signaling mechanisms that safeguard singularity during enterocyte polarization we generated knockout cells for the Cdc42 effector protein Par6A. Par6A loss results in the formation of multiple apical domains, similar to loss of Cdc42. In Par6A knockout cells, we find that active Cdc42 is more mobile at the apical membrane compared to control cells and that wild type Cdc42 is more diffusely localized throughout the cell, indicating that Par6A is required to restrict Cdc42 signaling. Par6A, Cdc42 and its GEF Tuba bind in a co-immunoprecipitation experiment and they partially colocalize at the apical membrane in polarized Ls174T:W4 cells, suggesting the formation of a trimeric complex. Indeed, in a rescue experiment using Par6A mutants, we show that the ability to establish this trimeric complex correlates with the ability to restore singularity in Par6A knockout cells. Together, these experiments therefore indicate that a Tuba/Cdc42/Par6A complex is required to ensure the formation of a single apical domain during enterocyte polarization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Polaridade Celular/genética , Proteínas do Citoesqueleto/química , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Estrutura Quaternária de Proteína , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/química
5.
Mol Cell Biol ; 38(12)2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29581186

RESUMO

PTEN is a tumor suppressor that is frequently lost in epithelial malignancies. A part of the tumor-suppressive properties of PTEN is attributed to its function in cell polarization and consequently its role in maintaining epithelial tissue integrity. However, surprisingly little is known about the function and regulation of PTEN during epithelial cell polarization. We used clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene disruption to delete PTEN in intestinal epithelial Ls174T:W4 cells, which upon differentiation form a microvillus-covered apical membrane (brush border) on a part of the cell cortex, independent of cell-cell junctions. We show that loss of PTEN results in the formation of a larger brush border that, in a fraction of the cells, even spans the entire plasma membrane, revealing that PTEN functions in the regulation of apical membrane size. Depletion of the phosphatase PTPL1 resulted in a similar defect. PTPL1 interacts with PTEN, and this interaction is necessary for apical membrane enrichment of PTEN. Importantly, phosphatase activity of PTPL1 is not required, indicating that PTPL1 functions as an anchor protein in this process. Our work thus demonstrates a novel function for PTEN during cell polarization in controlling apical membrane size and identifies PTPL1 as a critical apical membrane anchor for PTEN in this process.


Assuntos
Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Microvilosidades/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Células Epiteliais/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Microvilosidades/genética , Neoplasias/patologia , PTEN Fosfo-Hidrolase/genética
6.
Mol Cell Biol ; 37(7)2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28069739

RESUMO

Signaling by the small GTPase Cdc42 governs a diverse set of cellular processes that contribute to tissue morphogenesis. Since these processes often require highly localized signaling, Cdc42 activity must be clustered in order to prevent ectopic signaling. During cell polarization, apical Cdc42 signaling directs the positioning of the nascent apical membrane. However, the molecular mechanisms that drive Cdc42 clustering during polarity establishment are largely unknown. Here, we demonstrate that during cell polarization localized Cdc42 signaling is enabled via activity-dependent control of Cdc42 mobility. By performing photoconversion experiments, we show that inactive Cdc42-GDP is 30-fold more mobile than active Cdc42-GTP. This switch in apical mobility originates from a dual mechanism involving RhoGDI-mediated membrane dissociation of Cdc42-GDP and Tuba-mediated immobilization of Cdc42-GTP. Interference with either mechanism affects Cdc42 clustering and as a consequence impairs Cdc42-mediated apical membrane clustering. We therefore identify a molecular network, comprised of Cdc42, the guanine nucleotide exchange factor (GEF) Tuba, and RhoGDI, that enables differential diffusion of inactive and active Cdc42 and is required to establish localized Cdc42 signaling during enterocyte polarization.


Assuntos
Polaridade Celular , Enterócitos/citologia , Enterócitos/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Microvilosidades/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
7.
J Cell Biol ; 210(7): 1055-63, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26416959

RESUMO

During yeast cell polarization localization of the small GTPase, cell division control protein 42 homologue (Cdc42) is clustered to ensure the formation of a single bud. Here we show that the disease-associated flippase ATPase class I type 8b member 1 (ATP8B1) enables Cdc42 clustering during enterocyte polarization. Loss of this regulation results in increased apical membrane size with scattered apical recycling endosomes and permits the formation of more than one apical domain, resembling the singularity defect observed in yeast. Mechanistically, we show that to become apically clustered, Cdc42 requires the interaction between its polybasic region and negatively charged membrane lipids provided by ATP8B1. Disturbing this interaction, either by ATP8B1 depletion or by introduction of a Cdc42 mutant defective in lipid binding, increases Cdc42 mobility and results in apical membrane enlargement. Re-establishing Cdc42 clustering, by tethering it to the apical membrane or lowering its diffusion, restores normal apical membrane size in ATP8B1-depleted cells. We therefore conclude that singularity regulation by Cdc42 is conserved between yeast and human and that this regulation is required to maintain healthy tissue architecture.


Assuntos
Adenosina Trifosfatases/metabolismo , Polaridade Celular/fisiologia , Enterócitos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Adenosina Trifosfatases/genética , Animais , Linhagem Celular , Enterócitos/citologia , Humanos , Lipídeos de Membrana/genética , Camundongos , Proteínas de Transferência de Fosfolipídeos/genética , Proteína cdc42 de Ligação ao GTP/genética
8.
PLoS One ; 9(9): e106687, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25203140

RESUMO

Brush border formation during polarity establishment of intestinal epithelial cells is uniquely governed by the Rap2A GTPase, despite expression of the other highly similar Rap2 isoforms (Rap2B and Rap2C). We investigated the mechanisms of this remarkable specificity and found that Rap2C is spatially segregated from Rap2A signaling as it is not enriched at the apical membrane after polarization. In contrast, both Rap2A and Rap2B are similarly located at Rab11 positive apical recycling endosomes and inside the brush border. However, although Rap2B localizes similarly it is not equally activated as Rap2A during brush border formation. We reveal that the C-terminal hypervariable region allows selective activation of Rap2A, yet this selectivity does not originate from the known differential lipid modifications of this region. In conclusion, we demonstrate that Rap2 specificity during brush border formation is determined by two distinct mechanisms involving segregated localization and selective activation.


Assuntos
Enterócitos/ultraestrutura , Microvilosidades/metabolismo , Transdução de Sinais , Proteínas rap de Ligação ao GTP/metabolismo , Linhagem Celular , Enterócitos/citologia , Humanos , Isoformas de Proteínas/metabolismo , Transporte Proteico
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