RESUMO
Microbes in soil navigate interactions by recognizing kin, forming social groups, exhibiting antagonistic behavior, and engaging in competitive kin rivalry. Here, we investigated a novel phenomenon of self-growth suppression (sibling rivalry) observed in Bradyrhizobium diazoefficiens USDA 110. Swimming colonies of USDA 110 developed a distinct demarcation line and inter-colony zone when inoculated adjacent to each other. In addition to self, USDA 110 suppressed growth of other Bradyrhizobium strains and several other soil bacteria. We demonstrated that the phenomenon of sibling rivalry is due to growth suppression but not cell death. The cells in the inter-colony zone were culturable but have reduced respiratory activity, ATP levels and motility. The observed growth suppression was due to the presence of a diffusible effector compound. This effector was labile, preventing extraction, and identification, but it is unlikely a protein or a strong acid or base. This counterintuitive phenomenon of self-growth suppression suggests a strategic adaptation for conserving energy and resources in competitive soil environments. Bradyrhizobium's utilization of antagonism including self-growth suppression likely provides a competitive advantage for long-term success in soil ecosystems.
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We report herein that the anti-CD20 therapeutic antibody, rituximab, is rearranged into microclusters within the phagocytic synapse by macrophage Fcγ receptors (FcγR) during antibody-dependent cellular phagocytosis. These microclusters were observed to potently recruit Syk and to undergo rearrangements that were limited by the cytoskeleton of the target cell, with depolymerization of target-cell actin filaments leading to modest increases in phagocytic efficiency. Total internal reflection fluorescence analysis revealed that FcγR total phosphorylation, Syk phosphorylation, and Syk recruitment were enhanced when IgG-FcγR microclustering was enabled on fluid bilayers relative to immobile bilayers in a process that required Arp2/3. We conclude that on fluid surfaces, IgG-FcγR microclustering promotes signaling through Syk that is amplified by Arp2/3-driven actin rearrangements. Thus, the surface mobility of antigens bound by IgG shapes the signaling of FcγR with an unrecognized complexity beyond the zipper and trigger models of phagocytosis.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Fagocitose , Receptores de IgG , Transdução de Sinais , Quinase Syk , Receptores de IgG/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Quinase Syk/metabolismo , Animais , Camundongos , Rituximab/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/química , Fosforilação , HumanosRESUMO
The ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow-derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKO macrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling, and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNA-sequencing comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.
Assuntos
Receptor de Fator Estimulador de Colônias de Macrófagos , Ubiquitina , Camundongos , Animais , Ubiquitina/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Macrófagos/metabolismoRESUMO
Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole-genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole-genome screen and counter-screen strategy based on a pseudoviral platform that allowed identification of genes specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and vesicular stomatitis virus glycoprotein (VSV-G) mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed the identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein packaged into the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed fluorescent protein reporter upon entry reduced the number of gene hits and increase specificity for viral entry. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and low-density lipoprotein receptors, respectively, and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry-associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression. Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genes Virais , Receptores ViraisRESUMO
The DiversitabTM system produces target specific high titer fully human polyclonal IgG immunoglobulins from transchromosomic (Tc) bovines shown to be safe and effective against multiple virulent pathogens in animal studies and Phase 1, 2 and 3 human clinical trials. We describe the functional properties of a human monoclonal antibody (mAb), 38C2, identified from this platform, which recognizes recombinant H1 hemagglutinins (HAs) and induces appreciable antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Interestingly, 38C2 monoclonal antibody demonstrated no detectable neutralizing activity against H1N1 virus in both hemagglutination inhibition and virus neutralization assays. Nevertheless, this human monoclonal antibody induced appreciable ADCC against cells infected with multiple H1N1 strains. The HA-binding activity of 38C2 was also demonstrated in flow cytometry using Madin-Darby canine kidney cells infected with multiple influenza A H1N1 viruses. Through further investigation with the enzyme-linked immunosorbent assay involving the HA peptide array and 3-dimensional structural modeling, we demonstrated that 38C2 appears to target a conserved epitope located at the HA1 protomer interface of H1N1 influenza viruses. A novel mode of HA-binding and in vitro ADCC activity pave the way for further evaluation of 38C2 as a potential therapeutic agent to treat influenza virus infections in humans.
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Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Humanos , Animais , Cães , Bovinos , Epitopos , Anticorpos Monoclonais , Subunidades Proteicas , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Imunoglobulina G , Citotoxicidade Celular Dependente de AnticorposRESUMO
Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism.
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Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Esteróis/farmacologia , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/fisiologia , Colesterol/metabolismo , Clatrina/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Receptores da Transferrina/metabolismo , Esteróis/metabolismoRESUMO
Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.
Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Pinocitose/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Morfolinas/farmacologia , Fosfatidilinositóis/metabolismo , Pinocitose/efeitos dos fármacos , Células RAW 264.7RESUMO
The objective of this longitudinal cohort study was to determine the seroprevalence of antibodies to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in healthcare workers employed at healthcare settings in three rural counties in eastern South Dakota and western Minnesota from May 13, 2020, through December 22, 2020. Three blood draws were performed at five clinical sites and tested for the presence of antibodies against the SARS-CoV-2. Serum samples were tested for the presence of antibodies using a fluorescent microsphere immunoassay (FMIA), neutralization of SARS-CoV-2 spike-pseudotyped particles (SARS-CoV-2pp) assay, and serum virus neutralization (SVN) assay. The seroprevalence was determined to be 1/336 (0.29%) for samples collected from 5/13/20 to 7/13/20, 5/260 (1.92%) for samples collected from 8/13/20 to 9/25/20, and 35/235 (14.89%) for samples collected from 10/16/20 to 12/22/20. Eight of the 35 (22.8%) seropositive individuals identified in the final draw did not report a previous diagnosis with COVID-19. There was a high correlation (>90%) between the FMIA and virus neutralization assays. Each clinical site's seroprevalence was higher than the cumulative incidence for the general public in the respective county as reported by state public health agencies. As of December 2020, there was a high percentage (85%) of seronegative individuals in the study population.
Assuntos
Anticorpos Antivirais/sangue , COVID-19/epidemiologia , Pessoal de Saúde/estatística & dados numéricos , Serviços de Saúde Rural/estatística & dados numéricos , SARS-CoV-2/imunologia , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/diagnóstico , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Minnesota/epidemiologia , Testes de Neutralização , Estudos Soroepidemiológicos , South Dakota/epidemiologia , Adulto JovemRESUMO
Influenza remains a global health risk and challenge. Currently, neuraminidase (NA) inhibitors are extensively used to treat influenza, but their efficacy is compromised by the emergence of drug-resistant variants. Neutralizing antibodies targeting influenza A virus surface glycoproteins are critical components of influenza therapeutic agents and may provide alternative strategies to the existing countermeasures. However, the major hurdle for the extensive application of antibody therapies lies in the difficulty of generating nonimmunogenic antibodies in large quantities rapidly. Here, we report that one human monoclonal antibody (MAb), 53C10, isolated from transchromosomic (Tc) cattle exhibits potent neutralization and hemagglutination inhibition titers against different clades of H1N1 subtype influenza A viruses. In vitro selection of antibody escape mutants revealed that 53C10 recognizes a novel noncontinuous epitope in the hemagglutinin (HA) head domain involving three amino acid residues, glycine (G), serine (S), and glutamic acid (E) at positions 172, 207, and 212, respectively. The results of our experiments supported a critical role for substitution of arginine at position 207 (S207R) in mediating resistance to 53C10, while substitutions at either G172E or E212A did not alter antibody recognition and neutralization. The E212A mutation may provide structural stability for the epitope, while the substitution G172E probably compensates for loss of fitness introduced by S207R. Our results offer novel insights into the mechanism of action of MAb 53C10 and indicate its potential role in therapeutic treatment of H1 influenza virus infection in humans.IMPORTANCE Respiratory diseases caused by influenza viruses still pose a serious concern to global health, and neutralizing antibodies constitute a promising area of antiviral therapeutics. However, the potential application of antibodies is often hampered by the challenge in generating nonimmunogenic antibodies in large scale. In the present study, transchromosomic (Tc) cattle were used for the generation of nonimmunogenic monoclonal antibodies (MAbs), and characterization of such MAbs revealed one monoclonal antibody, 53C10, exhibiting a potent neutralization activity against H1N1 influenza viruses. Further characterization of the neutralization escape mutant generated using this MAb showed that three amino acid substitutions in the HA head domain contributed to the resistance. These findings emphasize the importance of Tc cattle in the production of nonimmunogenic MAbs and highlight the potential of MAb 53C10 in the therapeutic application against H1 influenza virus infection in humans.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Bovinos , Linhagem Celular , Humanos , Evasão da Resposta Imune , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A/genética , Modelos Moleculares , Mutação , Testes de Neutralização , Análise de Sequência de ProteínaRESUMO
The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 µm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Fagocitose/imunologia , Receptores de IgG/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Endossomos/imunologia , Humanos , Macrófagos/imunologia , Transdução de Sinais/imunologiaRESUMO
The identification of transcription factor binding sites and cis-regulatory motifs is a frontier whereupon the rules governing protein-DNA binding are being revealed. Here, we developed a new method (DEep Sequence and Shape mOtif or DESSO) for cis-regulatory motif prediction using deep neural networks and the binomial distribution model. DESSO outperformed existing tools, including DeepBind, in predicting motifs in 690 human ENCODE ChIP-sequencing datasets. Furthermore, the deep-learning framework of DESSO expanded motif discovery beyond the state-of-the-art by allowing the identification of known and new protein-protein-DNA tethering interactions in human transcription factors (TFs). Specifically, 61 putative tethering interactions were identified among the 100 TFs expressed in the K562 cell line. In this work, the power of DESSO was further expanded by integrating the detection of DNA shape features. We found that shape information has strong predictive power for TF-DNA binding and provides new putative shape motif information for human TFs. Thus, DESSO improves in the identification and structural analysis of TF binding sites, by integrating the complexities of DNA binding into a deep-learning framework.
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Biologia Computacional/estatística & dados numéricos , DNA/química , Aprendizado Profundo , Fatores de Transcrição/genética , Sítios de Ligação , Biologia Computacional/métodos , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Células K562 , Motivos de Nucleotídeos , Ligação Proteica , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismoRESUMO
The most highly studied endocytic pathway, clathrin-dependent endocytosis, mediates a wide range of fundamental processes including nutrient internalization, receptor recycling, and signal transduction. In order to model tissue specific and developmental aspects of this process, CRISPR/Cas9 genomic editing was utilized to fluorescently label the C-terminus of clathrin light chain A (CLTA) within the phenotypically normal, parental CRMi001-A human induced pluripotent stem cell line. Successfully edited cells were isolated by fluorescently activated cell sorting, remained karyotypically normal, and maintained their differentiation potential. This cell line facilitates imaging of endogenous clathrin trafficking within varied cell types.
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Sistemas CRISPR-Cas/genética , Clatrina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Linhagem Celular , Genes Reporter , HumanosRESUMO
Fluorescence resonance energy transfer (FRET) is a powerful tool to study macromolecular interactions such as protein-protein interactions (PPIs). Fluorescent protein (FP) fusions enable FRET-based PPI analysis of signaling pathways and molecular structure in living cells. Despite FRET's importance in PPI studies, FRET has seen limited use in quantifying the affinities of PPIs in living cells. Here, we have explored the relationship between FRET efficiency and PPI affinity over a wide range when expressed from a single plasmid system in Escherichia coli. Using live-cell microscopy and a set of 20 pairs of small interacting proteins, belonging to different structural folds and interaction affinities, we demonstrate that FRET efficiency can reliably measure the dissociation constant (KD ) over a range of mM to nM. A 10-fold increase in the interaction affinity results in 0.05 unit increase in FRET efficiency, providing sufficient resolution to quantify large affinity differences (> 10-fold) using live-cell FRET. This approach provides a rapid and simple strategy for assessment of PPI affinities over a wide range and will have utility for high-throughput analysis of protein interactions.
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Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/química , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas Luminescentes/metabolismo , Ligação ProteicaRESUMO
Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles. The key mechanistic question of how coat assembly produces membrane curvature has been studied with molecular and cellular structural biology approaches, without direct visualization of the process in living cells; resulting in two competing models for membrane bending. Here we use polarized total internal reflection fluorescence microscopy (pol-TIRF) combined with electron, atomic force, and super-resolution optical microscopy to measure membrane curvature during CME. Surprisingly, coat assembly accommodates membrane bending concurrent with or after the assembly of the clathrin lattice. Once curvature began, CME proceeded to scission with robust timing. Four color pol-TIRF showed that CALM accumulated at high levels during membrane bending, implicating its auxiliary role in curvature generation. We conclude that clathrin-coat assembly is versatile and that multiple membrane-bending trajectories likely reflect the energetics of coat assembly relative to competing forces.
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Membrana Celular/fisiologia , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Linhagem Celular , Humanos , Proteínas Monoméricas de Montagem de Clatrina/metabolismoRESUMO
Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. RESULTS: showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p<0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.
Assuntos
Autofagia , Vírus da Diarreia Viral Bovina Tipo 1/patogenicidade , Animais , Autofagia/efeitos dos fármacos , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Doenças dos Bovinos/virologia , Efeito Citopatogênico Viral , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/efeitos dos fármacos , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Cães , Células Madin Darby de Rim Canino , Sirolimo/farmacologia , Especificidade da Espécie , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Colony stimulating factor-1 receptor (CSF-1R), a receptor tyrosine kinase (RTK), is the master regulator of macrophage biology. CSF-1 can bind CSF-1R resulting in receptor activation and signalling essential for macrophage functions such as proliferation, differentiation, survival, polarization, phagocytosis, cytokine secretion, and motility. CSF-1R activation can only occur after the receptor is presented on the macrophage cell surface. This process is reliant upon the underlying macrophage receptor trafficking machinery. However, the mechanistic details governing this process are incompletely understood. C-terminal Eps15 Homology Domain-containing (EHD) proteins have recently emerged as key regulators of receptor trafficking but have not yet been studied in the context of macrophage CSF-1R signalling. In this manuscript, we utilize primary bone-marrow derived macrophages (BMDMs) to reveal a novel function of EHD1 as a regulator of CSF-1R abundance on the cell surface. We report that EHD1-knockout (EHD1-KO) macrophages cell surface and total CSF-1R levels are significantly decreased. The decline in CSF-1R levels corresponds with reduced downstream macrophage functions such as cell proliferation, migration, and spreading. In EHD1-KO macrophages, transport of newly synthesized CSF-1R to the macrophage cell surface was reduced and was associated with the shunting of the receptor to the lysosome, which resulted in receptor degradation. These findings reveal a novel and functionally important role for EHD1 in governing CSF-1R signalling via regulation of anterograde transport of CSF-1R to the macrophage cell surface.
Assuntos
Membrana Celular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Animais , Autoantígenos/metabolismo , Compartimento Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.