RESUMO
Immune checkpoint inhibition treatment using aPD-1 monoclonal antibodies is a promising cancer immunotherapy approach. However, its effect on tumor immunity is narrow, as most patients do not respond to the treatment or suffer from recurrence. We show that the crosstalk between conventional type I dendritic cells (cDC1) and T cells is essential for an effective aPD-1-mediated anti-tumor response. Accordingly, we developed a bispecific DC-T cell engager (BiCE), a reagent that facilitates physical interactions between PD-1+ T cells and cDC1. BiCE treatment promotes the formation of active dendritic/T cell crosstalk in the tumor and tumor-draining lymph nodes. In vivo, single-cell and physical interacting cell analysis demonstrates the distinct and superior immune reprogramming of the tumors and tumor-draining lymph nodes treated with BiCE as compared to conventional aPD-1 treatment. By bridging immune cells, BiCE potentiates cell circuits and communication pathways needed for effective anti-tumor immunity.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Anticorpos Biespecíficos/uso terapêutico , Células Dendríticas/imunologia , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologiaRESUMO
Microglia, the parenchymal brain macrophages of the central nervous system, have emerged as critical players in brain development and homeostasis. The immune functions of these cells, however, remain less well defined. We investigated contributions of microglia in a relapsing-remitting multiple sclerosis paradigm, experimental autoimmune encephalitis in C57BL/6 x SJL F1 mice. Fate mapping-assisted translatome profiling during the relapsing-remitting disease course revealed the potential of microglia to interact with T cells through antigen presentation, costimulation and coinhibition. Abundant microglia-T cell aggregates, as observed by histology and flow cytometry, supported the idea of functional interactions of microglia and T cells during remission, with a bias towards regulatory T cells. Finally, microglia-restricted interferon-γ receptor and major histocompatibility complex mutagenesis significantly affected the functionality of the regulatory T cell compartment in the diseased central nervous system and remission. Collectively, our data establish critical non-redundant cognate and cytokine-mediated interactions of microglia with CD4+ T cells during autoimmune neuroinflammation.
Assuntos
Encefalomielite Autoimune Experimental , Camundongos , Animais , Microglia , Linfócitos T Reguladores/patologia , Camundongos Endogâmicos C57BL , Comunicação CelularRESUMO
Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.
Assuntos
Tolerância a Antígenos Próprios , Linfócitos T , Timo , Animais , Camundongos , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Tolerância a Antígenos Próprios/imunologia , Tolerância a Antígenos Próprios/fisiologia , Linfócitos T/classificação , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Tecido Parenquimatoso , Células Musculares , Células Endócrinas , Cromatina , Transcrição Gênica , GrelinaRESUMO
Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.
Assuntos
Mitocôndrias , Saccharomyces cerevisiae , Potenciais da Membrana , Mitocôndrias/metabolismo , Transporte Biológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Bloom's syndrome (BLM) protein is a known nuclear helicase that is able to unwind DNA secondary structures such as G-quadruplexes (G4s). However, its role in the regulation of cytoplasmic processes that involve RNA G-quadruplexes (rG4s) has not been previously studied. Here, we demonstrate that BLM is recruited to stress granules (SGs), which are cytoplasmic biomolecular condensates composed of RNAs and RNA-binding proteins. BLM is enriched in SGs upon different stress conditions and in an rG4-dependent manner. Also, we show that BLM unwinds rG4s and acts as a negative regulator of SG formation. Altogether, our data expand the cellular activity of BLM and shed light on the function that helicases play in the dynamics of biomolecular condensates.
Assuntos
Quadruplex G , Grânulos de Estresse , Humanos , DNA/química , RecQ Helicases/metabolismo , RNA/genética , Grânulos de Estresse/metabolismoRESUMO
This "Best Practices in User Consultation" article is the result of a 2022 International Society for the Advancement of Cytometry (ISAC) membership survey that collected valuable insights from the shared research laboratory (SRL) community and of a group discussion at the CYTO 2022 workshop of the same name. One key takeaway is the importance of initiating a consultation at the outset of a flow cytometry project, particularly for trainees. This approach enables the improvement and standardization of every step, from planning experiments to interpreting data. This proactive approach effectively mitigates experimental bias and avoids superfluous trial and error, thereby conserving valuable time and resources. In addition to guidelines, the optimal approaches for user consultation specify communication channels, methods, and critical information, thereby establishing a structure for productive correspondence between SRL and users. This framework functions as an exemplar for establishing robust and autonomous collaborative relationships. User consultation adds value by providing researchers with the necessary information to conduct reproducible flow cytometry experiments that adhere to scientific rigor. By following the steps, instructions, and strategies outlined in these best practices, an SRL can readily tailor them to its own setting, establishing a personalized workflow and formalizing user consultation services. This article provides a pragmatic guide for improving the caliber and efficacy of flow cytometry research and aggregates the flow cytometry SRL community's collective knowledge regarding user consultation.
RESUMO
The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.
Assuntos
Laboratórios , Software , Citometria de FluxoRESUMO
Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E(2) (PGE(2)) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA(+) activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.
Assuntos
Actinas/imunologia , Medula Óssea/imunologia , Células-Tronco Hematopoéticas/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Actinas/genética , Animais , Medula Óssea/metabolismo , Medula Óssea/efeitos da radiação , Comunicação Celular/genética , Comunicação Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Sobrevivência Celular/efeitos da radiação , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Dinoprostona/biossíntese , Dinoprostona/imunologia , Raios gama , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos da radiação , Macrófagos/citologia , Macrófagos/efeitos da radiação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/efeitos da radiação , Camundongos , Monócitos/citologia , Monócitos/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiaçãoRESUMO
Marine viruses are the most abundant biological entity in the ocean and are considered as major evolutionary drivers of microbial life [C. A. Suttle, Nat. Rev. Microbiol. 5, 801-812 (2007)]. Yet, we lack quantitative approaches to assess their impact on the marine ecosystem. Here, we provide quantification of active viral infection in the bloom forming single-celled phytoplankton Emiliania huxleyi infected by the large virus EhV, using high-throughput single-molecule messenger RNA in situ hybridization (smFISH) of both virus and host transcripts. In natural samples, viral infection reached only 25% of the population despite synchronized bloom demise exposing the coexistence of infected and noninfected subpopulations. We prove that photosynthetically active cells chronically release viral particles through nonlytic infection and that viral-induced cell lysis can occur without viral release, thus challenging major assumptions regarding the life cycle of giant viruses. We could also assess active infection in cell aggregates linking viral infection and carbon export to the deep ocean [C. P. Laber et al., Nat. Microbiol. 3, 537-547 (2018)] and suggest a potential host defense strategy by enrichment of infected cells in sinking aggregates. Our approach can be applied to diverse marine microbial systems, opening a mechanistic dimension to the study of biotic interactions in the ocean.
Assuntos
Eutrofização , Vírus Gigantes/fisiologia , Haptófitas/virologia , Proteínas de Algas/genética , Interações Hospedeiro-Patógeno , Hibridização in Situ Fluorescente , Estágios do Ciclo de Vida , RNA Mensageiro/metabolismo , Água do Mar/microbiologia , Análise de Célula Única , Proteínas Virais/genética , Vírion/metabolismoRESUMO
The Hippo signaling pathway is a major regulator of organ size. In the liver, Hippo pathway deregulation promotes hyperplasia and hepatocellular carcinoma primarily through hyperactivation of its downstream effector, YAP. The LATS2 tumor suppressor is a core member of the Hippo pathway. A screen for LATS2-interacting proteins in liver-derived cells identified the transcription factor SREBP2, master regulator of cholesterol homeostasis. LATS2 down-regulation caused SREBP activation and accumulation of excessive cholesterol. Likewise, mice harboring liver-specific Lats2 conditional knockout (Lats2-CKO) displayed constitutive SREBP activation and overexpressed SREBP target genes and developed spontaneous fatty liver disease. Interestingly, the impact of LATS2 depletion on SREBP-mediated transcription was clearly distinct from that of YAP overexpression. When challenged with excess dietary cholesterol, Lats2-CKO mice manifested more severe liver damage than wild-type mice. Surprisingly, apoptosis, inflammation, and fibrosis were actually attenuated relative to wild-type mice, in association with impaired p53 activation. Subsequently, Lats2-CKO mice failed to recover effectively from cholesterol-induced damage upon return to a normal diet. Additionally, decreased LATS2 mRNA in association with increased SREBP target gene expression was observed in a subset of human nonalcoholic fatty liver disease cases. Together, these findings further highlight the tight links between tumor suppressors and metabolic homeostasis.
Assuntos
Fígado Gorduroso/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Colesterol na Dieta/farmacologia , Fígado Gorduroso/genética , Deleção de Genes , Regulação da Expressão Gênica/genética , Células Hep G2 , Homeostase/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos Knockout , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.
Assuntos
Quinase 3 da Glicogênio Sintase , Transdução de Sinais , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/farmacologia , FosforilaçãoRESUMO
p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in nontransformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-κB subunit. Moreover, it partly shifts p53's conformation and transcriptional output toward a state resembling cancer-associated p53 mutants and endows p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently down-regulated in breast cancer; we propose that such down-regulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.
Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Movimento Celular/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Via de Sinalização Hippo , Humanos , Mutação , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Fosforilação , Conformação Proteica , Proteína Supressora de Tumor p53/genéticaRESUMO
Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.
Assuntos
Apresentação de Antígeno , Antígenos/imunologia , Células Dendríticas/imunologia , Peptídeos/imunologia , Agregados Proteicos/imunologia , Animais , Antígenos/genética , Linhagem Celular , Células Dendríticas/metabolismo , Epitopos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Peptídeos/metabolismoRESUMO
Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age-related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage-induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)-κB kinase, leading to decreased cell survival. NF-κB activation induced TNF-α secretion and JNK activation to mediate death of senescent cells in a caspase- and JNK-dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.
Assuntos
Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , CamundongosRESUMO
Strategies employed by pathogenic enteric bacteria, such as Shigella, to subvert the host adaptive immunity are not well defined. Impairment of T lymphocyte chemotaxis by blockage of polarised edge formation has been reported upon Shigella infection. However, the functional impact of Shigella on T lymphocytes remains to be determined. Here, we show that Shigella modulates CD4+ T cell F-actin dynamics and increases cell cortical stiffness. The scanning ability of T lymphocytes when encountering antigen-presenting cells (APC) is subsequently impaired resulting in decreased cell-cell contacts (or conjugates) between the two cell types, as compared with non-infected T cells. In addition, the few conjugates established between the invaded T cells and APCs display no polarised delivery and accumulation of the T cell receptor to the contact zone characterising canonical immunological synapses. This is most likely due to the targeting of intracellular vesicular trafficking by the bacterial type III secretion system (T3SS) effectors IpaJ and VirA. The collective impact of these cellular reshapings by Shigella eventually results in T cell activation dampening. Altogether, these results highlight the combined action of T3SS effectors leading to T cell defects upon Shigella infection.
Assuntos
Citoesqueleto de Actina/metabolismo , Imunidade Adaptativa , Disenteria Bacilar/imunologia , Transporte Proteico/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Shigella/metabolismo , Actinas , Linhagem Celular , Complexo de Golgi , Humanos , Sinapses Imunológicas , Shigella/genética , Linfócitos T/imunologia , Sistemas de Secreção Tipo III/metabolismoRESUMO
Processing of external information by mammalian cells often involves seemingly redundant isoforms of signaling molecules and transcription factors. Understanding the functional relevance of coexpressed isoforms that respond to the same signal and control a shared set of genes is still limited. Here we show, using imaging of individual living mammalian cells, that the closely related transcription factors NFAT1 and NFAT4 possess distinct nuclear localization dynamics in response to cell stimulation. NFAT4 shows a fast response, with rapid stochastic bursts of nuclear localization. Burst frequency grows with signal level, while response amplitude is fixed. In contrast, NFAT1 has a slow, continuous response, and its amplitude increases with signal level. These diverse dynamical features observed for single cells are translated into different impulse response strategies at the cell population level. We suggest that dynamic response diversity of seemingly redundant genes can provide cells with enhanced capabilities of temporal information processing.
Assuntos
Núcleo Celular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Cálcio/fisiologia , Linhagem Celular , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Imunoglobulina E/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Análise de Célula Única , Imagem com Lapso de TempoRESUMO
Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C(+) monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2-TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1ß. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6C(hi) monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation.
Assuntos
Colite/patologia , Colo/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Multimerização Proteica , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/metabolismo , Animais , Antígenos Ly/análise , Colite/induzido quimicamente , Citocinas/metabolismo , Modelos Animais de Doenças , Imunofenotipagem , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Monócitos/química , Receptor 6 Toll-Like/metabolismoRESUMO
Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy. αSNAP, the adaptor molecule for the SNARE-priming enzyme N-ethylmaleimide-sensitive factor (NSF) is known to be crucial for intracellular membrane fusion processes, but its role in autophagy remains unclear. Here we demonstrated that knockdown of αSNAP leads to inhibition of autophagy, manifested by an accumulation of sealed autophagosomes located in close proximity to lysosomes but not fused with them. Under these conditions, moreover, association of both Atg9 and the autophagy-related SNARE protein syntaxin17 with the autophagosome remained unaffected. Finally, our results suggested that under starvation conditions, the levels of αSNAP, although low, are nevertheless sufficient to partially promote the SNARE priming required for autophagy. Taken together, these findings indicate that while autophagosomal-lysosomal membrane fusion is sensitive to inhibition of SNARE priming, the initial stages of autophagosome biogenesis and autophagosome expansion remain resistant to its loss.
Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas Qa-SNARE/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Proteínas de Transporte Vesicular/genética , Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Fusão de Membrana , Proteínas de Membrana/metabolismo , Biogênese de Organelas , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
The enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+ T, and CD8+ T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection.