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1.
J Cell Biol ; 222(7)2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37200023

RESUMO

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.


Assuntos
Actinas , Complexos Endossomais de Distribuição Requeridos para Transporte , Integrinas , Actinas/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Integrinas/genética , Integrinas/metabolismo , Transporte Proteico , Fosfolipídeos/química , Membrana Celular , Macrófagos , Células Dendríticas , Fibroblastos , Humanos , Conformação Proteica
2.
Nat Commun ; 13(1): 5371, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36100608

RESUMO

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.


Assuntos
Antígenos de Neoplasias , Linfoma de Células B , Animais , Antígenos de Neoplasias/metabolismo , Ácidos Graxos/metabolismo , Linfoma de Células B/genética , Camundongos , Palmitatos , Tetraspaninas/genética , Tetraspaninas/metabolismo
3.
J Nanobiotechnology ; 20(1): 64, 2022 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-35109860

RESUMO

BACKGROUND: While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics. RESULTS: To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy. CONCLUSIONS: A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.


Assuntos
Imunoconjugados , Neoplasias , Animais , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoconjugados/metabolismo , Imuno-Histoquímica , Camundongos , Neoplasias/diagnóstico por imagem , Distribuição Tecidual
4.
ACS Chem Biol ; 17(1): 240-251, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35000377

RESUMO

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.


Assuntos
Concentração de Íons de Hidrogênio , Imagem Óptica/métodos , Via Secretória , Adenosina Trifosfatases/antagonistas & inibidores , Brefeldina A/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/enzimologia , Complexo de Golgi/metabolismo , Humanos , Macrolídeos/farmacologia , Microscopia de Fluorescência/métodos , Transporte Proteico
5.
Blood Adv ; 6(7): 2254-2266, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35086136

RESUMO

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Linfoma Difuso de Grandes Células B , Proteômica , Antígenos de Neoplasias/genética , Linfócitos B/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Linfoma Difuso de Grandes Células B/patologia , Tetraspaninas/genética
6.
J Cell Sci ; 133(5)2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601617

RESUMO

Immune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-α, upon acute infection.


Assuntos
Citocinas/metabolismo , Inflamação/fisiopatologia , Via Secretória , Animais , Humanos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Modelos Biológicos , Transporte Proteico , Proteínas SNARE/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
7.
iScience ; 11: 160-177, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30612035

RESUMO

Antigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal "clickable" antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.

8.
J Mol Cell Biol ; 11(2): 144-157, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30016456

RESUMO

Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood. In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.


Assuntos
Endossomos/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Citocinas/metabolismo , Exocitose , Humanos , Lipopolissacarídeos , Macrófagos/metabolismo , Cultura Primária de Células , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Vesículas Transportadoras/metabolismo
9.
Small GTPases ; 10(4): 311-323, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-28489960

RESUMO

We recently identified a key role for SWAP70 as the tethering factor stabilizing F-actin filaments on the surface of phagosomes in human dendritic cells by interacting both with Rho-family GTPases and the lipid phosphatidylinositol (3,4)-bisphosphate. In this study, we aimed to investigate whether this role of SWAP70 was general among immune phagocytes. Our data reveal that SWAP70 is recruited to early phagosomes of macrophages and dendritic cells from both human and mouse. The putative inhibitor of SWAP70 sanguinarine blocked phagocytosis and F-actin polymerization, supporting a key role for SWAP70 in phagocytosis as demonstrated previously with knock-down. Moreover, SWAP70 was recently shown to sequester the F-actin severing protein cofilin and we investigated this relationship in phagocytosis. Our data show an increased activation of cellular cofilin upon siRNA knockdown of SWAP70. Finally, we explored whether SWAP70 would be recruited to the immune synapse between dendritic cells and T cells required for antigen presentation, as the formation of such synapses depends on F-actin. However, we observed that SWAP70 was depleted at immune synapses and specifically was recruited to phagosomes. Our data support an essential and specific role for SWAP70 in tethering and stabilizing F-actin to the phagosomal surface in a wide range of phagocytes.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Nucleares/metabolismo , Fagossomos/metabolismo , Animais , Benzofenantridinas/farmacologia , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Humanos , Isoquinolinas/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Sinapses/metabolismo
10.
Biosci Rep ; 38(6)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30463908

RESUMO

Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-α secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.


Assuntos
Células Dendríticas/imunologia , Hipóxia/imunologia , Inflamação/imunologia , MAP Quinase Quinase Quinases/imunologia , Proteínas Proto-Oncogênicas/imunologia , Fator de Necrose Tumoral alfa/imunologia , Hipóxia Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Hipóxia/genética , Inflamação/genética , MAP Quinase Quinase Quinases/genética , Monócitos/citologia , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor 4 Toll-Like/imunologia , Regulação para Cima
11.
FEBS Lett ; 592(9): 1535-1544, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29570778

RESUMO

Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin-6 (IL-6) in human dendritic cells. By comparing this to the total secreted IL-6, we estimate that secretory vesicles contain about 0.5-3 IL-6 molecules, but with a large spread among cells/donors. Moreover, IL-6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL-6 production. IL-6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs.


Assuntos
Células Dendríticas/citologia , Interleucina-6/metabolismo , Vesículas Secretórias/metabolismo , Membrana Celular/metabolismo , Humanos , Transporte Proteico
12.
Eur J Cell Biol ; 96(7): 705-714, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28688576

RESUMO

Cross-presentation of foreign antigen in major histocompatibility complex (MHC) class I by dendritic cells (DCs) requires activation of the NADPH-oxidase NOX2 complex. We recently showed that NOX2 is recruited to phagosomes by the SNARE protein VAMP8 where NOX2-produced reactive oxygen species (ROS) cause lipid oxidation and membrane disruption, promoting antigen translocation into the cytosol for cross-presentation. In this study, we extend these findings by showing that VAMP8 is also involved in NOX2 trafficking to endosomes. Moreover, we demonstrate in both human and mouse DCs that absence of VAMP8 leads to decreased ROS production, lipid peroxidation and antigen translocation, and that this impairs cross-presentation. In contrast, knockdown of VAMP8 did not affect recruitment of MHC class I and the transporter associated with antigen processing 1 (TAP1) to phagosomes, although surface levels of MHC class I were reduced. Thus, in addition to a secretory role, VAMP8-mediates trafficking of NOX2 to endosomes and phagosomes and this promotes induction of cytolytic T cell immune responses.


Assuntos
Apresentação de Antígeno/genética , Células Dendríticas/imunologia , NADPH Oxidase 2/genética , Proteínas R-SNARE/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Apresentação de Antígeno/imunologia , Membrana Celular/genética , Membrana Celular/imunologia , Endossomos/genética , Endossomos/imunologia , Genes MHC Classe I/imunologia , Humanos , Peroxidação de Lipídeos , Camundongos , NADPH Oxidase 2/imunologia , Fagossomos/genética , Fagossomos/imunologia , Proteínas R-SNARE/imunologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Citotóxicos/imunologia
13.
Elife ; 62017 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-28524818

RESUMO

SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution.


Assuntos
Células Dendríticas/fisiologia , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Proteínas Qa-SNARE/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Interleucina-6/metabolismo , Transporte Proteico
14.
J Cell Sci ; 130(7): 1285-1298, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28202687

RESUMO

In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.


Assuntos
Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Fagossomos/metabolismo , Compartimento Celular , Membrana Celular/metabolismo , Grupo dos Citocromos b/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Membranas Intracelulares/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Modelos Biológicos , NADPH Oxidase 2 , Oxirredução , Fosfatidilinositóis/metabolismo , Proteínas Qa-SNARE , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo
15.
Cell Rep ; 17(6): 1518-1531, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27806292

RESUMO

Actin plays a critical role during the early stages of pathogenic microbe internalization by immune cells. In this study, we identified a key mechanism of actin filament tethering and stabilization to the surface of phagosomes in human dendritic cells. We found that the actin-binding protein SWAP70 is specifically recruited to nascent phagosomes by binding to the lipid phosphatidylinositol (3,4)-bisphosphate. Multi-color super-resolution stimulated emission depletion (STED) microscopy revealed that the actin cage surrounding early phagosomes is formed by multiple concentric rings containing SWAP70. SWAP70 colocalized with and stimulated activation of RAC1, a known activator of actin polymerization, on phagosomes. Genetic ablation of SWAP70 impaired actin polymerization around phagosomes and resulted in a phagocytic defect. These data show a key role for SWAP70 as a scaffold for tethering the peripheral actin cage to phagosomes.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Nucleares/metabolismo , Fagocitose , Actinas/metabolismo , Células Dendríticas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Polimerização , Proteínas rac1 de Ligação ao GTP/metabolismo
16.
Sci Rep ; 6: 22064, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26907999

RESUMO

Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer.


Assuntos
Apresentação Cruzada , Células Dendríticas/imunologia , Doença Granulomatosa Crônica/imunologia , Peroxidação de Lipídeos/imunologia , Glicoproteínas de Membrana/imunologia , NADPH Oxidases/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Apresentação de Antígeno , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Endossomos/imunologia , Endossomos/metabolismo , Sequestradores de Radicais Livres/farmacologia , Expressão Gênica , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/patologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Jurkat , Peroxidação de Lipídeos/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/genética , Fármacos Fotossensibilizantes/farmacologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , alfa-Tocoferol/farmacologia
17.
PLoS One ; 7(7): e41039, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22815903

RESUMO

The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by ß1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of ß1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.


Assuntos
Polaridade Celular/fisiologia , Regulação da Expressão Gênica , Quinases Ativadas por p21/metabolismo , Animais , Biotinilação , Linhagem Celular , Junções Célula-Matriz , Cães , Matriz Extracelular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Integrina beta1/metabolismo , Microscopia Confocal/métodos , Modelos Biológicos , Fenótipo , RNA Interferente Pequeno/metabolismo , Tripsina/química
18.
Mol Biol Cell ; 22(12): 2031-41, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21508319

RESUMO

Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.


Assuntos
Caderinas/metabolismo , Túbulos Renais/embriologia , Rim/embriologia , Junções Aderentes/metabolismo , Animais , Caderinas/genética , Caderinas/imunologia , Adesão Celular , Agregação Celular , Diferenciação Celular , Linhagem Celular , Polaridade Celular , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Rim/citologia , Rim/metabolismo , Túbulos Renais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA
19.
J Biol Chem ; 286(12): 10834-46, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21278252

RESUMO

To generate and maintain epithelial cell polarity, specific sorting of proteins into vesicles destined for the apical and basolateral domain is required. Syntaxin 3 and 4 are apical and basolateral SNARE proteins important for the specificity of vesicle fusion at the apical and basolateral plasma membrane domains, respectively, but how these proteins are specifically targeted to these domains themselves is unclear. Munc18/SM proteins are potential regulators of this process. Like syntaxins, they are crucial for exocytosis and vesicle fusion. However, how munc18c and syntaxin 4 regulate the function of each other is unclear. Here, we investigated the requirement of syntaxin 4 in the delivery of basolateral membrane and secretory proteins, the basolateral targeting of syntaxin 4, and the role of munc18c in this targeting. Depletion of syntaxin 4 resulted in significant reduction of basolateral targeting, suggesting no compensation by other syntaxin forms. Mutational analysis identified amino acids Leu-25 and to a lesser extent Val-26 as essential for correct localization of syntaxin 4. Recently, it was shown that the N-terminal peptide of syntaxin 4 is involved in binding to munc18c. A mutation in this region that affects munc18c binding shows that munc18c binding is required for stabilization of syntaxin 4 at the plasma membrane but not for its correct targeting. We conclude that the N terminus serves two functions in membrane targeting. First, it harbors the sorting motif, which targets syntaxin 4 basolaterally in a munc18c-independent manner and second, it allows for munc18c binding, which stabilizes the protein in a munc18c-dependent manner.


Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Membrana Celular/genética , Cães , Proteínas Munc18/genética , Mutação de Sentido Incorreto , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estabilidade Proteica , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismo
20.
Methods Mol Biol ; 440: 171-86, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18369945

RESUMO

The plasma membrane of epithelial cells has two physically separated membrane domains. This membrane polarization is essential for the function of epithelial cells. It has been well established that different plasma membrane syntaxin forms are expressed in epithelial cells. In addition, these syntaxin forms can have a polarized localization, suggesting that they may play a direct role in the specificity of polarized membrane delivery. To determine the mechanism of the polarized syntaxin localization, we have made several chimeras of syntaxin 3 and 4. This allowed us to identify the protein sequences involved in this polarized localization. Using this technique, we showed that targeting information of syntaxin 3 and 4 is located in the first 30 amino acids.


Assuntos
Bioensaio/métodos , Polaridade Celular , Células Epiteliais/metabolismo , Proteínas Qa-SNARE/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Linhagem Celular , Clusterina/metabolismo , Cães , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Vetores Genéticos , Microscopia Confocal , Transporte Proteico , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transdução Genética
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