RESUMO
Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.
Assuntos
Células Dendríticas , Vesículas Extracelulares , Monócitos , Humanos , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Monócitos/metabolismo , Monócitos/imunologia , Monócitos/microbiologia , Citometria de Fluxo/métodos , Endocitose , Leveduras/metabolismo , Saccharomyces cerevisiae/metabolismo , Células CultivadasRESUMO
Oil-in-water emulsion is a system with extensive applications in foods, cosmetics and coating industries, and it could also be designed into an artificial lipid droplet in recent works. However, the insights into the biophysical dynamic behaviors of such artificial lipid droplets are lacking. Here, we reveal an enzymatic reaction triggered endocytosis-like behavior in the oil-in-water emulsion lipid droplets. A thermodynamically favored recruitment of lipases onto the membrane of the droplets is demonstrated. We confirm that the hydrolysis of tributyrin by lipases can decrease the interfacial tension and increase the compressive force on the membrane, which are the two main driving forces for triggering the endocytosis-like behavior. The endocytosis-like behavior induced various emerging functionalities of the lipid droplets, including proteins, DNA or inorganic particles being efficiently sequestered into the oil droplet with reversible release as well as enhanced cascade enzymatic reaction. Overall, our studies are expected to open up a way to functionalize oil-in-water emulsions capable of life-inspired behaviors and tackle emerging challenges in bottom-up synthetic biology, revealing the unknown dynamic behaviors of lipid droplets in living organisms.
Assuntos
Emulsões , Endocitose , Lipase , Gotículas Lipídicas , Óleos , Triglicerídeos , Água , Emulsões/química , Lipase/metabolismo , Lipase/química , Água/química , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Triglicerídeos/química , Triglicerídeos/metabolismo , Óleos/química , HidróliseRESUMO
Purpose: Castration Resistant Prostate Cancer (CRPC) is characterized by poor prognosis and limited therapeutic options. AgNPs functionalized with glucose (G-AgNPs) were observed cytotoxic to CRPC cell lines (PC-3 and Du-145) and not LNCaP. This study aims to evaluate AgNPs and G-AgNPs' uptake mechanisms in these cells and understand their role in the selective effect against CRPC cells. Methods: Uptake of AgNPs and G-AgNPs was assessed through transmission electron microscopy (TEM). A microRNA (miRNAs) analysis approach was used to uncover the main molecular differences responsible for the endocytic mechanisms' regulation. Caveolin (Cav) 1 and 2 mRNA and protein levels were assessed in the three cell lines. Caveolae-dependent endocytosis was inhibited with genistein or siCav1- and siCav2- in PC-3 and Du-145 and resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration. Caveolae-dependent endocytosis was induced with Cav1+ and Cav2+ plasmids in LNCaP, resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration and TEM to assess their location. Results: AgNPs and G-AgNPs were not uptaked by LNCaP. miRNA analysis revealed 37 upregulated and 90 downregulated miRNAs. Functional enrichment analysis of miRNAs' targets resulted in enrichment of terms related to endocytosis and caveolae. We observed that Cav1 and Cav2 are not expressed in LNCaP. Inhibiting caveolae-dependent endocytosis in Du-145 and PC-3 led to a significative reduction of cytotoxic capacity of AgNPs and G-AgNPs and induction of caveolae-dependent endocytosis in LNCaP lead to a significative increase as well as their uptake by cells. Conclusion: This study shows the potential of these AgNPs as a new therapeutic approach directed to CRPC patients, uncovers caveolae-dependent endocytosis as the uptake mechanism of these AgNPs and highlights deregulation of Cav1 and Cav2 expression as a key difference in hormone sensitive and resistant PCa cells which may be responsible for drug resistance.
Assuntos
Cavéolas , Caveolina 1 , Endocitose , Nanopartículas Metálicas , MicroRNAs , Neoplasias de Próstata Resistentes à Castração , Prata , Masculino , Humanos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Cavéolas/metabolismo , Cavéolas/efeitos dos fármacos , Prata/química , Prata/farmacologia , Prata/farmacocinética , Caveolina 1/metabolismo , Caveolina 1/genética , Nanopartículas Metálicas/química , Linhagem Celular Tumoral , MicroRNAs/metabolismo , MicroRNAs/genética , Sobrevivência Celular/efeitos dos fármacos , Caveolina 2/metabolismo , Caveolina 2/genética , Antineoplásicos/farmacologia , Células PC-3RESUMO
Introduction: Immunogenicity, the unwanted immune response triggered by therapeutic antibodies, poses significant challenges in biotherapeutic development. This response can lead to the production of anti-drug antibodies, potentially compromising the efficacy and safety of treatments. The internalization of therapeutic antibodies into dendritic cells (DCs) is a critical factor influencing immunogenicity. Using monoclonal antibodies, with differences in non-specific cellular uptake, as tools to explore the impact on the overall risk of immunogenicity, this study explores how internalization influences peptide presentation and subsequently T cell activation. Materials and methods: To investigate the impact of antibody internalization on immunogenicity, untargeted toolantibodies with engineered positive or negative charge patches were utilized. Immature monocyte-derived DCs (moDCs), known for their physiologically relevant high endocytic activity, were employed for internalization assays, while mature moDCs were used for MHC-II associated peptide proteomics (MAPPs) assays. In addition to the lysosomal accumulation and peptide presentation, subsequent CD4+ T cell activation has been assessed. Consequently, a known CD4+ T cell epitope from ovalbumin was inserted into the tool antibodies to evaluate T cell activation on a single, shared epitope. Results: Antibodies with positive charge patches exhibited higher rates of lysosomal accumulation and epitope presentation compared to those with negative charge patches or neutral surface charge. Furthermore, a direct correlation between internalization rate and presentation on MHC-II molecules could be established. To explore the link between internalization, peptide presentation and CD4+ T cell activation, tool antibodies containing the same OVA epitope were used. Previous observations were not altered by the insertion of the OVA epitope and ultimately, an enhanced CD4+ T cell response correlated with increased internalization in DCs and peptide presentation. Discussion: These findings demonstrate that the biophysical properties of therapeutic antibodies, particularly surface charge, play a crucial role in their internalization into DCs. Antibodies internalized faster and processed by DCs, are also more prone to be presented on their surface leading to a higher risk of triggering an immune response. These insights underscore the importance of considering antibody surface charge and other properties that enhance cellular accumulation during the preclinical development of biotherapeutics to mitigate immunogenicity risks.
Assuntos
Apresentação de Antígeno , Células Dendríticas , Ativação Linfocitária , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Apresentação de Antígeno/imunologia , Ativação Linfocitária/imunologia , Anticorpos Monoclonais/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Fatores de Risco , Endocitose/imunologia , Ovalbumina/imunologiaRESUMO
In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.
Assuntos
Esclerose Lateral Amiotrófica , Modelos Animais de Doenças , Endocitose , Neurônios Motores , Vesículas Sinápticas , Animais , Camundongos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Diafragma/inervação , Diafragma/metabolismo , Diafragma/fisiopatologia , Endocitose/fisiologia , Corantes Fluorescentes/metabolismo , Imidazóis/farmacologia , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Terminações Nervosas/metabolismo , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Transmissão Sináptica/fisiologia , Transmissão Sináptica/genética , Vesículas Sinápticas/metabolismoRESUMO
The excessive inflammation caused by the prolonged activation of Toll-like receptor 4 (TLR4) and its downstream signaling pathways leads to sepsis. CD14-mediated endocytosis of TLR4 is the key step to control the amount of TLR4 on cell membrane and the activity of downstream pathways. The actin cytoskeleton is necessary for receptor-mediated endocytosis, but its role in TLR4 endocytosis remains elusive. Here we show that Tropomodulin 1 (Tmod1), an actin capping protein, inhibited lipopolysaccharide (LPS)-induced TLR4 endocytosis and intracellular trafficking in macrophages. Thus it resulted in increased surface TLR4 and the upregulation of myeloid differentiation factor 88 (MyD88)-dependent pathway and the downregulation of TIR domain-containing adaptor-inducing interferon-ß (TRIF)-dependent pathway, leading to the enhanced secretion of inflammatory cytokines, such as TNF-α and IL-6, and the reduced secretion of cytokines, such as IFN-ß. Macrophages deficient with Tmod1 relieved the inflammatory response in LPS-induced acute lung injury mouse model. Mechanistically, Tmod1 negatively regulated LPS-induced TLR4 endocytosis and inflammatory response through modulating the activity of CD14/Syk/PLCγ2/IP3/Ca2+ signaling pathway, the reorganization of actin cytoskeleton, and the membrane tension. Therefore, Tmod1 is a key regulator of inflammatory response and immune functions in macrophages and may be a potential target for the treatment of excessive inflammation and sepsis.
Assuntos
Endocitose , Inflamação , Lipopolissacarídeos , Macrófagos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptor 4 Toll-Like , Tropomodulina , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Células RAW 264.7 , Receptor 4 Toll-Like/metabolismo , Tropomodulina/metabolismo , Tropomodulina/genéticaRESUMO
The extensive use of plastic products has exacerbated micro/nanoplastic (MPs/NPs) pollution in the atmosphere, increasing the incidence of respiratory diseases and lung cancer. This study investigates the uptake and cytotoxicity mechanisms of polystyrene (PS) NPs in human lung epithelial cells. Transcriptional analysis revealed significant changes in cell adhesion pathways following PS-NPs exposure. Integrin α5ß1-mediated endocytosis was identified as a key promoter of PS-NPs entry into lung epithelial cells. Overexpression of integrin α5ß1 enhanced PS-NPs internalization, exacerbating mitochondrial Ca2+ dysfunction and depolarization, which induced reactive oxygen species (ROS) production. Mitochondrial dysfunction triggered by PS-NPs led to oxidative damage, inflammation, DNA damage, and necrosis, contributing to lung diseases. This study elucidates the molecular mechanism by which integrin α5ß1 facilitates PS-NPs internalization and enhances its cytotoxicity, offering new insights into potential therapeutic targets for microplastic-induced lung diseases.
Assuntos
Endocitose , Pneumopatias , Poliestirenos , Humanos , Poliestirenos/toxicidade , Pneumopatias/induzido quimicamente , Integrina alfa5beta1/metabolismo , Microplásticos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Nanopartículas/toxicidadeRESUMO
Hematopoietic stem cells (HSCs) have emerged as one of the most therapeutically significant adult stem cells, paving way for a range of novel curative regimens over decades. HSCs are transplanted, either directly or post restorative genetic engineering in order to repopulate a healthy hematopoietic homeostasis in patients with disorders affecting the blood and immune cells. Despite being an extensively studied system, the maintenance and expansion of functional HSCs ex vivo remains a major bottleneck. The challenge primarily stems from difficulties in reproducing HSC self-renewal divisions and gradual depletion of stemness characters, in vitro. Refining the in vitro culture can be particularly beneficial in the case of cord blood HSCs (CB-HSCs), as inadequate numbers in a single umbilical cord limits its therapeutic potential. In recent years, molecular dissection of HSC stemness has significantly improved in vitro hematopoietic stem and progenitor cells (HSPCs) culture. Despite such significant progress, lacunae exist in fully understanding all the underlying mechanisms and their interplay active in bona fide HSCs, and how it transforms when cells proliferate in culture. A new groundbreaking study titled "MYCT1 controls environmental sensing in human haematopoietic stem cells", published in Nature in June 2024, sheds light on this complex field. Through a series of experiments, including knock-down, overexpression, single-cell RNA sequencing, and transplantation, the study identifies a previously unknown role of the MYC target 1 (MYCT1) protein in HSC maintenance. This protein acts as a crucial regulator of human HSCs, with high expression in primitive HSCs and subsequently downregulated during ex vivo culture. The study reveals that MYCT1 plays a vital role in moderating endocytosis and environmental sensing in HSCs, processes thereby essential for maintaining HSC stemness and function. This commentary will discuss the implications of the new findings for cord blood expansion in cell therapies and HSPC culture for gene therapy applications, providing valuable insights for the field of hematopoietic regenerative medicine.
Assuntos
Endocitose , Terapia Genética , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Diferenciação Celular , AnimaisRESUMO
Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe. End4p directly transmits force in CME by binding to both the membrane (through the AP180 N-terminal homology [ANTH] domain) and F-actin (through the talin-HIP1/R/Sla2p actin-tethering C-terminal homology [THATCH] domain). We show that 7 pN force is required for stable binding between THATCH and F-actin. We also characterized a domain in End4p, Rend (rod domain in End4p), that resembles R12 of talin. Membrane localization of Rend primes the binding of THATCH to F-actin, and force-induced unfolding of Rend at 15 pN terminates the transmission of force. We show that the mechanical properties (mechanical stability, unfolding extension, hysteresis) of Rend and THATCH are tuned to form a circuit for the initiation, transmission, and termination of force between the actin cytoskeleton and membrane. The mechanical circuit by Rend and THATCH may be conserved and coopted evolutionarily in cell adhesion complexes.
Assuntos
Actinas , Clatrina , Endocitose , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Endocitose/fisiologia , Schizosaccharomyces/metabolismo , Clatrina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Actinas/metabolismo , Domínios Proteicos , Citoesqueleto de Actina/metabolismo , Ligação Proteica , Membrana Celular/metabolismoRESUMO
Secondary envelopment of the human cytomegalovirus (HCMV) is a critical but not well-understood process that takes place at the cytoplasmic viral assembly complex (cVAC) where nucleocapsids acquire their envelope by budding into cellular membranes containing viral glycoproteins. Previous studies presented controversial results regarding the composition of the viral envelope, suggesting trans-Golgi and endosomal origins, as well as intersections with the exosomal and endocytic pathways. Here, we investigated the role of endocytic membranes for the secondary envelopment of HCMV by using wheat germ agglutinin (WGA) pulse labeling to label glycoproteins at the plasma membrane and to follow their trafficking during HCMV infection by light microscopy and transmission electron microscopy (TEM). WGA labeled different membrane compartments within the cVAC, including early endosomes, multivesicular bodies, trans-Golgi, and recycling endosomes. Furthermore, TEM analysis showed that almost 90% of capsids undergoing secondary envelopment and 50% of enveloped capsids were WGA-positive within 90 min. Our data reveal extensive remodeling of the endocytic compartment in the late stage of HCMV infection, where the endocytic compartment provides an optimized environment for virion morphogenesis and serves as the primary membrane source for secondary envelopment. Furthermore, we show that secondary envelopment is a rapid process in which endocytosed membranes are transported from the plasma membrane to the cVAC within minutes to be utilized by capsids for envelopment.
Assuntos
Membrana Celular , Citomegalovirus , Endocitose , Montagem de Vírus , Humanos , Citomegalovirus/fisiologia , Citomegalovirus/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Envelope Viral/metabolismo , Endossomos/virologia , Endossomos/metabolismo , Aglutininas do Germe de Trigo/metabolismo , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/metabolismoRESUMO
Essentially all plasma membrane proteins are glycosylated, and their activity is regulated by tuning their cell surface dynamics. This is achieved by glycan-binding proteins of the galectin family that either retain glycoproteins within lattices or drive their endocytic uptake via the clathrin-independent glycolipid-lectin (GL-Lect) mechanism. Here, we have used immunofluorescence-based assays to analyze how lattice and GL-Lect mechanisms affect the internalization of the cell adhesion and migration glycoprotein α5ß1 integrin. In retinal pigment epithelial (RPE-1) cells, internalized α5ß1 integrin is found in small peripheral endosomes under unperturbed conditions. Pharmacological compounds were used to competitively inhibit one of the galectin family members, galectin-3 (Gal3), or to inhibit the expression of glycosphingolipids, both of which are the fabric of the GL-Lect mechanism. We found that under acute inhibition conditions, endocytic uptake of α5ß1 integrin was strongly reduced, in agreement with previous studies on the GL-Lect driven internalization of the protein. In contrast, upon prolonged inhibitor treatment, the uptake of α5ß1 integrin was increased, and the protein was now internalized by alternative pathways into large perinuclear endosomes. Our findings suggest that under these prolonged inhibitor treatment conditions, α5ß1 integrin containing galectin lattices are dissociated, leading to an altered endocytic compartmentalization.
Assuntos
Endocitose , Galectina 3 , Integrina alfa5beta1 , Humanos , Galectina 3/metabolismo , Integrina alfa5beta1/metabolismo , Linhagem Celular , Endossomos/metabolismo , Adesão Celular , Galectinas/metabolismo , Proteínas SanguíneasRESUMO
The dynamic process of membrane shaping and remodeling plays a vital role in cellular functions, with proteins and cellular membranes interacting intricately to adapt to various cellular needs and environmental cues. Ubiquitination-a posttranslational modification-was shown to be essential in regulating membrane structure and shape. It influences virtually all pathways relying on cellular membranes, such as endocytosis and autophagy by directing protein degradation, sorting, and oligomerization. Ubiquitin is mostly known as a protein modifier; however, it was reported that ubiquitin and ubiquitin-like proteins can associate directly with lipids, affecting membrane curvature and dynamics. In this review, we summarize some of the current knowledge on ubiquitin-mediated membrane remodeling in the context of endocytosis, autophagy, and ER-phagy.
Assuntos
Membrana Celular , Ubiquitina , Ubiquitinação , Ubiquitina/metabolismo , Humanos , Membrana Celular/metabolismo , Autofagia , Endocitose , Animais , Retículo Endoplasmático/metabolismoRESUMO
Polarized exocytosis induced by local Cdc42 GTPase activity results in membrane flows that deplete low-mobility membrane-associated proteins. A reaction-diffusion particle model comprising Cdc42 positive feedback activation, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis shows that flow-induced depletion of low mobility GAPs promotes polarization. We modified Cdc42 mobility in Schizosaccharomyces pombe by replacing its prenylation site with 1, 2 or 3 repeats of the Rit C-terminal membrane-binding domain (ritC), yielding alleles with progressively lower mobility and increased flow-coupling. While Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC are inviable, in agreement with model's predictions. Deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells, verifying the model's prediction that GAP deletion increases Cdc42 activity at the expense of polarization. Our work demonstrates how membrane flows are an integral part of Cdc42-driven pattern formation and require Cdc42-GTP to turn over faster than the surface on which it forms.
Assuntos
Membrana Celular , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteína cdc42 de Ligação ao GTP , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Membrana Celular/metabolismo , Polaridade Celular , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Forma Celular , Exocitose/fisiologia , EndocitoseRESUMO
The CTLA-4 and PD-1 checkpoints control immune responses and are key targets in immunotherapy. Both pathways are connected via a cis interaction between CD80 and PD-L1, the ligands for CTLA-4 and PD-1, respectively. This cis interaction prevents PD-1-PD-L1 binding but is reversed by CTLA-4 trans-endocytosis of CD80. However, how CTLA-4 selectively removes CD80, but not PD-L1, is unclear. Here, we show CTLA-4-CD80 interactions are unimpeded by PD-L1 and that CTLA-4 binding with CD80 does not displace PD-L1 per se. Rather, both rigidity and bivalency of CTLA-4 molecules are required to orientate CD80 such that PD-L1 interactions are no longer permissible. Moreover, soluble CTLA-4 released PD-L1 only at specific expression levels of CD80 and PD-L1, whereas CTLA-4 trans-endocytosis released PD-L1 in all conditions. These data show that PD-L1 release from CD80 is driven by orientation and bivalent cross-linking of membrane proteins and that trans-endocytosis of CD80 efficiently promotes PD-L1 availability.
Assuntos
Antígeno B7-1 , Antígeno B7-H1 , Antígeno CTLA-4 , Ligação Proteica , Antígeno B7-1/metabolismo , Antígeno CTLA-4/metabolismo , Antígeno B7-H1/metabolismo , Humanos , Animais , Endocitose , CamundongosRESUMO
Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion.
Assuntos
Proteínas de Drosophila , Endossomos , Transporte Proteico , Proteína Wnt1 , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Asas de Animais/metabolismo , Proteína Wnt1/metabolismo , Proteína Wnt1/genéticaRESUMO
Overcoming intestinal epithelial barriers to enhance bioavailability is a major challenge for oral delivery systems. Desirable nanocarriers should simultaneously exhibit rapid mucus penetration and efficient epithelial uptake; however, they two generally require contradictory structural properties. Herein, we proposed a strategy to construct multiperformance nanoparticles by modifying the rigidity of amphiphilic nanostructures originating from soy polypeptides (SPNPs), where its ability to overcome multibarriers was examined from both in vitro and in vivo, using curcumin (CUR) as a model cargo. Low-rigidity SPNPs showed higher affinity to mucin and were prone to getting stuck in the mucus layer. When they reached epithelial cells, they tended to be endocytosed through the clathrin and macropinocytosis pathways and further transferred to lysosomes, showing severe degradation and lower transport of CUR. Increased particle rigidity generally improved the absorption of CUR, with medium-rigidity SPNPs bloomed maximum plasma concentration of CUR by 80.62-fold and showed the highest oral bioavailability. Results from monocultured and cocultured cell models demonstrated that medium-rigidity SPNPs were least influenced by the mucus layer and changes in rigidity significantly influenced the endocytosis and intracellular fate of SPNPs. Those with higher rigidity preferred to be endocytosed via a caveolae-mediated pathway and trafficked to the ER and Golgi, facilitating their whole transcytosis, and avoiding intracellular metabolism. Moreover, rigidity modulation efficiently induces the reversible opening of intercellular tight junctions, which synergistically improves the transport of CUR into blood circulation. This study suggested that rigidity regulation on food originated amphiphilic peptides could overcome multiple physiological barriers, showing great potential as natural building block toward oral delivery.
Assuntos
Disponibilidade Biológica , Curcumina , Muco , Curcumina/química , Curcumina/farmacocinética , Curcumina/farmacologia , Curcumina/metabolismo , Animais , Humanos , Muco/metabolismo , Muco/química , Nanopartículas/química , Nanopartículas/metabolismo , Portadores de Fármacos/química , Células CACO-2 , Tamanho da Partícula , Endocitose , Ratos Sprague-Dawley , Administração Oral , Peptídeos/química , Peptídeos/metabolismo , Camundongos , MasculinoRESUMO
Mitochondria, the dynamic organelles responsible for energy production and cellular metabolism, have the metabolic function of extracting energy from nutrients and synthesizing crucial metabolites. Nevertheless, recent research unveils that intercellular mitochondrial transfer by tunneling nanotubes, tumor microtubes, gap junction intercellular communication, extracellular vesicles, endocytosis and cell fusion may regulate mitochondrial function within recipient cells, potentially contributing to disease treatment, such as nonalcoholic steatohepatitis, glioblastoma, ischemic stroke, bladder cancer and neurodegenerative diseases. This review introduces the principal approaches to intercellular mitochondrial transfer and examines its role in various diseases. Furthermore, we provide a comprehensive overview of the inhibitors and activators of intercellular mitochondrial transfer, offering a unique perspective to illustrate the relationship between intercellular mitochondrial transfer and diseases.
Assuntos
Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Animais , Comunicação Celular , Vesículas Extracelulares/metabolismo , Transporte Biológico , Endocitose/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapiaRESUMO
We previously reported that leukotriene B4 (LTB4) contained in Trichomonas vaginalis-derived secretory products (TvSP) play an essential role in interleukin-8 (IL-8) production in human mast cell line (HMC-1 cells) via LTB4 receptor (BLT)-mediated Nuclear Factor-kappa B (NF-кB) activation. Dynamin, a GTPase, has been known to be involved in endocytosis of receptors for signaling of production of cytokine or chemokines. In the present study, we investigated the role of dynamin-mediated BLT1 endocytosis in TvSP-induced IL-8 production. When HMC-1 cells were transfected with BLT1 or BLT2 siRNA, TvSP-induced IL-8 production was significantly inhibited compared with that in cells transfected with control siRNA. In addition, pretreatment of HMC-1 cells with a dynamin inhibitor (Dynasore) reduced IL-8 production induced by TvSP or LTB4. TvSP- or LTB4- induced phosphorylation of NF-кB was also attenuated by pretreatment with Dynasore. After exposing HMC-1 cells to TvSP or LTB4, BLT1 was translocated from the intracellular compartments to the plasma membrane within 30 min. At 60 min after stimulation with TvSP or LTB4, BLT1 remigrated from the cell surface to intracellular areas. Pretreatment of HMC-1 cells with dynamin-2 siRNA blocked internalization of BLT1 induced by TvSP or LTB4. Co-immunoprecipitation experiments revealed that dynamin-2 strongly interacted with BLT1 60 min after stimulation with TvSP or LTB4. These results suggest that T. vaginalis-secreted LTB4 induces IL-8 production in HMC-1 cells via dynamin 2-mediated endocytosis of BLT1 and phosphorylation of NF-кB.
Assuntos
Dinamina II , Endocitose , Interleucina-8 , Receptores do Leucotrieno B4 , Trichomonas vaginalis , Humanos , Interleucina-8/metabolismo , Interleucina-8/genética , Receptores do Leucotrieno B4/metabolismo , Receptores do Leucotrieno B4/genética , Endocitose/efeitos dos fármacos , Dinamina II/metabolismo , Dinamina II/genética , Linhagem Celular , Trichomonas vaginalis/metabolismo , Leucotrieno B4/metabolismo , Mastócitos/metabolismo , Mastócitos/imunologia , NF-kappa B/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.