RESUMO
The plasma membrane tetraspan molecule MS4A4A is selectively expressed by macrophage-lineage cells, but its function is unknown. Here we report that MS4A4A was restricted to murine and human mononuclear phagocytes and was induced during monocyte-to-macrophage differentiation in the presence of interleukin 4 or dexamethasone. Human MS4A4A was co-expressed with M2/M2-like molecules in subsets of normal tissue-resident macrophages, infiltrating macrophages from inflamed synovium and tumor-associated macrophages. MS4A4A interacted and colocalized with the ß-glucan receptor dectin-1 in lipid rafts. In response to dectin-1 ligands, Ms4a4a-deficient macrophages showed defective signaling and defective production of effector molecules. In experimental models of tumor progression and metastasis, Ms4a4a deficiency in macrophages had no impact on primary tumor growth, but was essential for dectin-1-mediated activation of macrophages and natural killer (NK) cell-mediated metastasis control. Thus, MS4A4A is a tetraspan molecule selectively expressed in macrophages during differentiation and polarization, essential for dectin-1-dependent activation of NK cell-mediated resistance to metastasis.
Assuntos
Células Matadoras Naturais/imunologia , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Metástase Neoplásica/imunologia , Neoplasias/imunologia , Animais , Diferenciação Celular/imunologia , Linhagem da Célula , Dexametasona/farmacologia , Humanos , Interleucina-4/metabolismo , Ativação Linfocitária/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Metástase Neoplásica/prevenção & controle , Neoplasias/patologiaRESUMO
Two-photon intravital microscopy (2P-IVM) has become a widely used technique to study cell-to-cell interactions in living organisms. Four-dimensional imaging data obtained via 2P-IVM are classically analyzed by performing automated cell tracking, a procedure that computes the trajectories followed by each cell. However, technical artifacts, such as brightness shifts, the presence of autofluorescent objects, and channel crosstalking, affect the specificity of imaging channels for the cells of interest, thus hampering cell detection. Recently, machine learning has been applied to overcome a variety of obstacles in biomedical imaging. However, existing methods are not tailored for the specific problems of intravital imaging of immune cells. Moreover, results are highly dependent on the quality of the annotations provided by the user. In this study, we developed CANCOL, a tool that facilitates the application of machine learning for automated tracking of immune cells in 2P-IVM. CANCOL guides the user during the annotation of specific objects that are problematic for cell tracking when not properly annotated. Then, it computes a virtual colocalization channel that is specific for the cells of interest. We validated the use of CANCOL on challenging 2P-IVM videos from murine organs, obtaining a significant improvement in the accuracy of automated tracking while reducing the time required for manual track curation.
Assuntos
Comunicação Celular , Microscopia Intravital , Animais , Artefatos , Rastreamento de Células , Computadores , Microscopia Intravital/métodos , CamundongosRESUMO
Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/genética , Proteólise , alfa 1-Antitripsina/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Calnexina/genética , Calnexina/metabolismo , Retículo Endoplasmático/genética , Endossomos/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , alfa 1-Antitripsina/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Innate immunity is involved in regulating inflammatory and tissue repair responses to injury. In particular, humoral innate immunity plays functions related to wound clearance from tissue debris, and regulation of macrophage and stromal cell activities. PTX3, a component of humoral innate immunity, orchestrates tissue repair by interacting with plasminogen and fibrin. Fluid-phase molecules of innate immunity interact with elements of the extracellular matrix, and some of the latter display opsonic activity against certain bacterial species. Thus, recognition of extracellular matrix and microbial components is a recurrent theme in the humoral arm of the innate immune system.
Assuntos
Bactérias/imunologia , Proteína C-Reativa/metabolismo , Matriz Extracelular/metabolismo , Imunidade Humoral , Imunidade Inata , Proteínas do Tecido Nervoso/metabolismo , Animais , Humanos , CicatrizaçãoRESUMO
Human colorectal cancer (CRC) is a frequent neoplasia in Western countries, and its metastatic progression is a major cause of cancer-related death. In search of specific molecules upregulated in CRC, with possible clinical relevance, we performed a differential gene-profiling analysis in surgery-derived CRC samples and adjacent uninvolved intestinal mucosa. The chemokine CX3CL1 and its specific receptor CX3CR1 were significantly upregulated in tumors. Higher expression of CX3CL1 and CX3CR1 was confirmed by immunohistochemistry in 100 CRC tumor samples (stages I-III). Unexpectedly, high immune scores of CX3CL1 did not correlate with the density of tumor-infiltrating CD3(+) T cells or CD68(+) macrophages. Coexpression of ligand and receptor by tumor cells (axis-positive tumors) significantly associated with longer disease-free (p = 0.01) and disease-specific survival (p = 0.001). Conversely, axis-negative tumors (with low expression of both ligand and receptor) had increased risk of tumor relapse (p = 0.02), and increased likelihood of metachronous metastasis (p = 0.001), including after stage adjustment (p = 0.006). Transduction of CX3CL1 and CX3CR1 in CRC tumor cell lines induced cell aggregation that strongly inhibited in vitro migration in chemotaxis assays. In a mouse model of spleen-liver metastases, cancer dissemination to liver was dramatically reduced in CX3CL1-CX3CR1-expressing tumors, and ligand-receptor interaction was confirmed in cancer cells in vivo by fluorescence resonance energy transfer analysis. In conclusion, tumoral expression of the CX3CL1-CX3CR1 chemokine axis functions as a retention factor, increasing homotypic cell adhesion and limiting tumor spreading to metastatic sites. Lack or low levels of expression of CX3CL1-CX3CR1 by tumor cells identifies a group of CRC patients at increased risk of metastatic progression.
Assuntos
Quimiocina CX3CL1/biossíntese , Neoplasias Colorretais/patologia , Invasividade Neoplásica/patologia , Receptores de Quimiocinas/biossíntese , Animais , Receptor 1 de Quimiocina CX3C , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Intervalo Livre de Doença , Feminino , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Reação em Cadeia da Polimerase , Prognóstico , Modelos de Riscos Proporcionais , TranscriptomaRESUMO
We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within =/~20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/°C) and low uncertainty (±0.3 °C). Gold nanostars are =/~3 and =/~100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia.
Assuntos
Ouro/química , Nanopartículas de Magnetita/química , Nanopartículas Metálicas/química , Temperatura , Fenômenos Ópticos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
ABSTRACT: Surgery, burns or surgery-free accident are leading causes of scars with altered tissue consistency, a reduced degree of motion and pain. Autologous fat grafting can dramatically improve tissue consistency and elasticity but less frequently results in the reduction of pain. Therefore, we analyzed different cell populations present within the adipose tissue to be engrafted and correlated them with the reduction of pain after surgery. Here, we identify a population of CD3 - CD4 - CD304 + cells present in grafted adipose tissue, whose abundance highly correlates with pain improvement shortly after surgery ( r2 = 0.7243****) as well as persistently over time (3 months later: r2 = 0.6277****, 1 year later: r2 = 0.5346***, and 4 years later: r2 = 0.5223***). These cells are characterized by the absence of the hematopoietic marker CD45, whereas they express CD90 and CD34, which characterize mesenchymal stem cells (MSCs); the concomitant presence of CD10 and CD73 in the plasma membrane supports a function of these cells in pain reduction. We deduce that the enrichment of this adipose tissue-derived MSC subset could enhance the therapeutic properties of adipose grafts and ameliorate localized pain syndromes.
Assuntos
Células-Tronco Mesenquimais , Humanos , Tecido Adiposo/transplante , Dor/metabolismo , Síndrome , Diferenciação Celular , Células CultivadasRESUMO
Cell migration is a pivotal biological process, whose dysregulation is found in many diseases including inflammation and cancer. Advances in microscopy technologies allow now to study cell migration in vitro, within engineered microenvironments that resemble in vivo conditions. However, to capture an entire 3D migration chamber for extended periods of time and with high temporal resolution, images are generally acquired with low resolution, which poses a challenge for data analysis. Indeed, cell detection and tracking are hampered due to the large pixel size (i.e., cell diameter down to 2 pixels), the possible low signal-to-noise ratio, and distortions in the cell shape due to changes in the z-axis position. Although fluorescent staining can be used to facilitate cell detection, it may alter cell behavior and it may suffer from fluorescence loss over time (photobleaching).Here we describe a protocol that employs an established deep learning method (U-NET), to specifically convert transmitted light (TL) signal from unlabeled cells imaged with low resolution to a fluorescent-like signal (class 1 probability). We demonstrate its application to study cancer cell migration, obtaining a significant improvement in tracking accuracy, while not suffering from photobleaching. This is reflected in the possibility of tracking cells for three-fold longer periods of time. To facilitate the application of the protocol we provide WID-U, an open-source plugin for FIJI and Imaris imaging software, the training dataset used in this paper, and the code to train the network for custom experimental settings.
RESUMO
The endoplasmic reticulum (ER) is an organelle of nucleated cells that produces proteins, lipids and oligosaccharides. ER volume and activity are increased upon induction of unfolded protein responses (UPR) and are reduced upon activation of ER-phagy programs. A specialized domain of the ER, the nuclear envelope (NE), protects the cell genome with two juxtaposed lipid bilayers, the inner and outer nuclear membranes (INM and ONM) separated by the perinuclear space (PNS). Here we report that expansion of the mammalian ER upon homeostatic perturbations results in TMX4 reductase-driven disassembly of the LINC complexes connecting INM and ONM and in ONM swelling. The physiologic distance between ONM and INM is restored, upon resolution of the ER stress, by asymmetric autophagy of the NE, which involves the LC3 lipidation machinery, the autophagy receptor SEC62 and the direct capture of ONM-derived vesicles by degradative LAMP1/RAB7-positive endolysosomes in a catabolic pathway mechanistically defined as micro-ONM-phagy.
Assuntos
Estresse do Retículo Endoplasmático , Membrana Nuclear , Animais , Estresse do Retículo Endoplasmático/genética , Autofagia , Resposta a Proteínas não Dobradas , Retículo Endoplasmático , MamíferosRESUMO
Secretory immunoglobulin A (SIgA) interaction with commensal bacteria conditions microbiota composition and function. However, mechanisms regulating reciprocal control of microbiota and SIgA are not defined. Bacteria-derived adenosine triphosphate (ATP) limits T follicular helper (Tfh) cells in the Peyer's patches (PPs) via P2X7 receptor (P2X7R) and thereby SIgA generation. Here we show that hydrolysis of extracellular ATP (eATP) by apyrase results in amplification of the SIgA repertoire. The enhanced breadth of SIgA in mice colonized with apyrase-releasing Escherichia coli influences topographical distribution of bacteria and expression of genes involved in metabolic versus immune functions in the intestinal epithelium. SIgA-mediated conditioning of bacteria and enterocyte function is reflected by differences in nutrient absorption in mice colonized with apyrase-expressing bacteria. Apyrase-induced SIgA improves intestinal homeostasis and attenuates barrier impairment and susceptibility to infection by enteric pathogens in antibiotic-induced dysbiosis. Therefore, amplification of SIgA by apyrase can be leveraged to restore intestinal fitness in dysbiotic conditions.
Assuntos
Apirase , Imunoglobulina A Secretora , Trifosfato de Adenosina/metabolismo , Animais , Bactérias/metabolismo , Homeostase , Imunoglobulina A Secretora/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Camundongos , Nódulos Linfáticos AgregadosRESUMO
During melanoma metastasis, tumor cells originating in the skin migrate via lymphatic vessels to the sentinel lymph node (sLN). This process facilitates tumor cell spread across the body. Here, we characterized the innate inflammatory response to melanoma in the metastatic microenvironment of the sLN. We found that macrophages located in the subcapsular sinus (SS) produced protumoral IL1α after recognition of tumoral antigens. Moreover, we confirmed that the elimination of LN macrophages or the administration of an IL1α-specific blocking antibody reduced metastatic spread. To understand the mechanism of action of IL1α in the context of the sLN microenvironment, we applied single-cell RNA sequencing to microdissected metastases obtained from animals treated with the IL1α-specific blocking antibody. Among the different pathways affected, we identified STAT3 as one of the main targets of IL1α signaling in metastatic tumor cells. Moreover, we found that the antitumoral effect of the anti-IL1α was not mediated by lymphocytes because Il1r1 knockout mice did not show significant differences in metastasis growth. Finally, we found a synergistic antimetastatic effect of the combination of IL1α blockade and STAT3 inhibition with stattic, highlighting a new immunotherapy approach to preventing melanoma metastasis.
Assuntos
Vasos Linfáticos , Melanoma , Linfonodo Sentinela , Neoplasias Cutâneas , Animais , Camundongos , Biópsia de Linfonodo Sentinela , Linfonodo Sentinela/patologia , Metástase Linfática/patologia , Melanoma/patologia , Macrófagos/metabolismo , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Linfonodos/patologia , Neoplasias Cutâneas/patologia , Microambiente TumoralRESUMO
The coronavirus spike glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus spike proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors. These mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and betacoronaviruses, including animal coronaviruses WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses showed that the fusion peptide-specific mAbs bound with different modalities to a cryptic epitope hidden in prefusion stabilized spike, which became exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais , Anticorpos Antivirais , Anticorpos Amplamente Neutralizantes , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Humanos , Peptídeos/imunologia , Ligação Proteica , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Clinical progression of tauopathies may result from transcellular propagation of pathogenic Tau seeds with the possible involvement of extracellular vesicles (EVs) as transport vectors. We established a cell model for investigating EV delivery of proteins, since the mechanism regulating EV cargo delivery to recipient cells is poorly understood. In our cell model, EVs are readily internalized and accumulate in degradative organelles (DOs). We then show for the first time that in this acidic compartment, profibrillogenic Tau delivered by EVs interacts with Tau expressed by the recipient cells and cause its accumulation by a process that involves the participation of autophagy. Thus, the degradative compartment of cells may represent the subcellular site initiating a cascade of events resulting in early hallmarks of tauopathies. These are characterized by seeded Tau accumulation, pathology-associated epitopes, DO stress, and cytotoxicity. The involvement of autophagy to this process and the relative accessibility of the degradative pathway for extracellular agents, support possible modes of intervention to slow down the progression of neurodegeneration.
Assuntos
Vesículas Extracelulares/metabolismo , Organelas/metabolismo , Tauopatias , Proteínas tau/fisiologia , Animais , Linhagem Celular , Camundongos , Células-Tronco Multipotentes , Tauopatias/metabolismo , Tauopatias/patologiaRESUMO
Endolysosomal compartments maintain cellular fitness by clearing dysfunctional organelles and proteins from cells. Modulation of their activity offers therapeutic opportunities. Quantification of cargo delivery to and/or accumulation within endolysosomes is instrumental for characterizing lysosome-driven pathways at the molecular level and monitoring consequences of genetic or environmental modifications. Here we introduce LysoQuant, a deep learning approach for segmentation and classification of fluorescence images capturing cargo delivery within endolysosomes for clearance. LysoQuant is trained for unbiased and rapid recognition with human-level accuracy, and the pipeline informs on a series of quantitative parameters such as endolysosome number, size, shape, position within cells, and occupancy, which report on activity of lysosome-driven pathways. In our selected examples, LysoQuant successfully determines the magnitude of mechanistically distinct catabolic pathways that ensure lysosomal clearance of a model organelle, the endoplasmic reticulum, and of a model protein, polymerogenic ATZ. It does so with accuracy and velocity compatible with those of high-throughput analyses.
Assuntos
Biologia Computacional/métodos , Organelas/metabolismo , Transporte Proteico/fisiologia , Aprendizado Profundo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Peptídeos/metabolismo , Dobramento de Proteína , Proteínas/metabolismoRESUMO
Acidic pH occurs in acute wounds progressing to healing as consequence of a cell metabolic adaptation in response to injury-induced tissue hypoperfusion. In tumours, high metabolic rate leads to acidosis affecting cancer progression. Acidic pH affects activities of remodelling cells in vitro. The pH measurement predicts healing in pathological wounds and success of surgical treatment of burns and chronic ulcers. However, current methods are limited to skin surface or based on detection of fluorescence intensity of specific sensitive probes that suffer of microenvironment factors. Herein, we ascertained relevance in vivo of cell metabolic adaptation in skin repair by interfering with anaerobic glycolysis. Moreover, a custom-designed skin imaging chamber, 2-Photon microscopy (2PM), fluorescence lifetime imaging (FLIM) and data mapping analyses were used to correlate maps of glycolytic activity in vivo as measurement of NADH intrinsic lifetime with areas of hypoxia and acidification in models of skin injury and cancer. The method was challenged by measuring the NADH profile by interfering with anaerobic glycolysis and oxidative phosphorylation in the mitochondrial respiratory chain. Therefore, intravital NADH FLIM represents a tool for investigating cell metabolic adaptation occurring in wounds, as well as the relationship between cell metabolism and cancer.
Assuntos
Dermoscopia/métodos , Microscopia de Fluorescência/métodos , Neoplasias , Cicatrização , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Microambiente Celular , Feminino , Glicólise , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , NAD/metabolismo , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. Currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. Secretory immunoglobulin A (SIgA) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. In this study, we describe the prophylactic activity of orally delivered recombinant SIgA generated from two human monoclonal antibodies (CAA1 and CCG4) isolated for their reactivity against the flagellar-capping protein FliD, which is essential for bacteria motility and highly conserved across Campylobacter species associated with severe enteritis. In an immunocompetent weaned mouse model, a single oral administration of FliD-reactive SIgA CAA1 or CCG4 at 2 h before infection significantly enhances Campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (PMN) cells infiltration in the cecum lamina propria. Our data indicate that the prophylactic activity of CAA1 and CCG4 is not only dependent on the specificity to FliD but also on the use of the SIgA format, as the immunoglobulin G (IgG) versions of the same antibodies did not confer a comparable protective effect. Our work emphasizes the potential of FliD as a target for the development of vaccines and supports the concept that orally administered FliD-reactive SIgA can be developed to prevent or mitigate the severity of Campylobacter infections as well as the development of post-infection syndromes.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Infecções por Campylobacter/imunologia , Campylobacter/fisiologia , Gastroenterite/imunologia , Imunoterapia/métodos , Mucosa Intestinal/imunologia , Neutrófilos/imunologia , Animais , Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais/metabolismo , Modelos Animais de Doenças , Resistência à Doença , Feminino , Humanos , Imunoglobulina A/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The endoplasmic reticulum (ER) produces about 40% of the nucleated cell's proteome. ER size and content in molecular chaperones increase upon physiologic and pathologic stresses on activation of unfolded protein responses (UPR). On stress resolution, the mammalian ER is remodeled to pre-stress, physiologic size and function on activation of the LC3-binding activity of the translocon component SEC62. This elicits recov-ER-phagy, i.e., the delivery of the excess ER generated during the phase of stress to endolysosomes (EL) for clearance. Here, ultrastructural and genetic analyses reveal that recov-ER-phagy entails the LC3 lipidation machinery and proceeds via piecemeal micro-ER-phagy, where RAB7/LAMP1-positive EL directly engulf excess ER in processes that rely on the Endosomal Sorting Complex Required for Transport (ESCRT)-III component CHMP4B and the accessory AAA+ ATPase VPS4A. Thus, ESCRT-III-driven micro-ER-phagy emerges as a key catabolic pathway activated to remodel the mammalian ER on recovery from ER stress.
Assuntos
Autofagia/fisiologia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Resposta a Proteínas não Dobradas , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Autofagossomos/metabolismo , Endossomos/metabolismo , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas SNARE/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Aim: To design lympho-targeted nanocarriers with the capacity to enhance the activity of associated drugs/antigens whose target is within the lymphatic system. Materials & methods: Inulin (INU)-based nanocapsules (NCs), negatively charged and positively charged chitosan NCs were prepared by the solvent displacement techniques. The NCs were produced in two sizes: small (70 nm) and medium (170-250 nm). Results:In vitro results indicated that small NCs interacted more efficiently with dendritic cells than the larger ones. The study of the NCs biodistribution in mice, using 3D reconstruction of the popliteal lymph node, showed that small INU NCs have the greatest access and uniform accumulation in different subsets of resident immune cells. Conclusion: Small and negatively charged INU NCs have a potential as lympho-targeted antigen/drug nanocarriers.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanocápsulas/química , Polímeros/química , Células Cultivadas , Quitosana/química , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/ultraestrutura , Linfonodos/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanocápsulas/ultraestruturaRESUMO
Cancer stem cells (CSCs) contribute to disease progression and treatment failure in human cancers. The balance among self-renewal, differentiation, and senescence determines the expansion or progressive exhaustion of CSCs. Targeting these processes might lead to novel anticancer therapies. Here, we uncover a novel link between BRD4, mitochondrial dynamics, and self-renewal of prostate CSCs. Targeting BRD4 by genetic knockdown or chemical inhibitors blocked mitochondrial fission and caused CSC exhaustion and loss of tumorigenic capability. Depletion of CSCs occurred in multiple prostate cancer models, indicating a common vulnerability and dependency on mitochondrial dynamics. These effects depended on rewiring of the BRD4-driven transcription and repression of mitochondrial fission factor (Mff). Knockdown of Mff reproduced the effects of BRD4 inhibition, whereas ectopic Mff expression rescued prostate CSCs from exhaustion. This novel concept of targeting mitochondrial plasticity in CSCs through BRD4 inhibition provides a new paradigm for developing more effective treatment strategies for prostate cancer.