RESUMO
Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.
Assuntos
Metaloendopeptidases/metabolismo , Zinco , Animais , GTP Fosfo-Hidrolases/metabolismo , Homeostase , Metalochaperonas/metabolismo , Metaloproteínas/genética , Camundongos , Peixe-Zebra/metabolismo , Zinco/metabolismoRESUMO
Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.
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Clostridioides difficile , Infecções por Clostridium , Compostos Férricos , Interações entre Hospedeiro e Microrganismos , Ferro , Organelas , Animais , Camundongos , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/imunologia , Clostridioides difficile/metabolismo , Infecções por Clostridium/imunologia , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Ferro/metabolismo , Organelas/metabolismo , Homeostase , Compostos Férricos/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Modelos Animais de Doenças , Complexo Antígeno L1 Leucocitário/metabolismo , Viabilidade Microbiana , Inflamação/metabolismo , Inflamação/microbiologia , Intestinos/metabolismo , Intestinos/microbiologiaRESUMO
Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.
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Vesículas Extracelulares , Orthoreovirus Mamífero 3 , Orthoreovirus de Mamíferos , Orthoreovirus , Reoviridae , Animais , Camundongos , Humanos , Células CACO-2 , Reoviridae/genética , Orthoreovirus Mamífero 3/genética , MamíferosRESUMO
Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-lysyl oxidase inhibitor ß-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) mice and mice heterozygous for a missense mutation in Fbn1 (Fbn1C1041G/+) for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis.
Assuntos
Aminopropionitrilo , Modelos Animais de Doenças , Fibrilina-1 , Síndrome de Marfan , Nervo Óptico , Proteína-Lisina 6-Oxidase , Animais , Camundongos , Adipocinas , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/genética , Aminopropionitrilo/farmacologia , Tecido Elástico/patologia , Tecido Elástico/metabolismo , Tecido Elástico/ultraestrutura , Fibrilina-1/genética , Fibrilinas/metabolismo , Glaucoma/patologia , Pressão Intraocular , Síndrome de Marfan/patologia , Síndrome de Marfan/complicações , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Nervo Óptico/efeitos dos fármacos , Proteína-Lisina 6-Oxidase/metabolismo , Proteína-Lisina 6-Oxidase/antagonistas & inibidoresRESUMO
BACKGROUND & AIMS: Elements of field cancerization, including atrophic gastritis, metaplasia, and dysplasia, promote gastric cancer development in association with chronic inflammation. However, it remains unclear how stroma changes during carcinogenesis and how the stroma contributes to progression of gastric preneoplasia. Here we investigated heterogeneity of fibroblasts, one of the most important elements in the stroma, and their roles in neoplastic transformation of metaplasia. METHODS: We used single-cell transcriptomics to evaluate the cellular heterogeneity of mucosal cells from patients with gastric cancer. Tissue sections from the same cohort and tissue microarrays were used to identify the geographical distribution of distinct fibroblast subsets. We further evaluated the role of fibroblasts from pathologic mucosa in dysplastic progression of metaplastic cells using patient-derived metaplastic gastroids and fibroblasts. RESULTS: We identified 4 subsets of fibroblasts within stromal cells defined by the differential expression of PDGFRA, FBLN2, ACTA2, or PDGFRB. Each subset was distributed distinctively throughout stomach tissues with different proportions at each pathologic stage. The PDGFRα+ subset expanded in metaplasia and cancer compared with normal, maintaining a close proximity with the epithelial compartment. Co-culture of metaplasia- or cancer-derived fibroblasts with gastroids showing the characteristics of spasmolytic polypeptide-expressing metaplasia-induced disordered growth, loss of metaplastic markers, and increases in markers of dysplasia. Culture of metaplastic gastroids with conditioned media from metaplasia- or cancer-derived fibroblasts also promoted dysplastic transition. CONCLUSIONS: These findings indicate that fibroblast associations with metaplastic epithelial cells can facilitate direct transition of metaplastic spasmolytic polypeptide-expressing metaplasia cell lineages into dysplastic lineages.
Assuntos
Mucosa Gástrica , Neoplasias Gástricas , Humanos , Mucosa Gástrica/patologia , Neoplasias Gástricas/patologia , Hiperplasia , Metaplasia/patologia , Fibroblastos/metabolismoRESUMO
In polarized MDCK cells, disruption of the tyrosine-based YXXΦ basolateral trafficking motif (Y156A) in the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG), results in its apical mistrafficking and transformation in vivo. However, the mechanisms underlying these dramatic effects are unknown. Using a doxycycline-inducible system in 3D Matrigel cultures, we now show that induction of Y156A EREG in fully formed MDCK cysts results in direct and complete delivery of mutant EREG to the apical cell surface. Within 3 days of induction, ectopic lumens were detected in mutant, but not wild-type, EREG-expressing cysts. Of note, these structures resembled histological features found in subcutaneous xenografts of mutant EREG-expressing MDCK cells. These ectopic lumens formed de novo rather than budding from the central lumen and depended on metalloprotease-mediated cleavage of EREG and subsequent EGFR activity. Moreover, the most frequent EREG mutation in human cancer (R147stop) resulted in its apical mistrafficking in engineered MDCK cells. Thus, induction of EREG apical mistrafficking is sufficient to disrupt selective aspects of polarity of a preformed polarized epithelium. This article has an associated First Person interview with the first author of the paper.
Assuntos
Receptores ErbB , Transdução de Sinais , Epirregulina/genética , Epirregulina/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , FosforilaçãoRESUMO
Intestinal enterocytes have an elaborate apical membrane of actin-rich protrusions known as microvilli. The organization of microvilli is orchestrated by the intermicrovillar adhesion complex (IMAC), which connects the distal tips of adjacent microvilli. The IMAC is composed of CDHR2 and CDHR5 as well as the scaffolding proteins USH1C, ANKS4B, and Myosin 7b (MYO7B). To create an IMAC, cells must transport the proteins to the apical membrane. Myosin 5b (MYO5B) is a molecular motor that traffics ion transporters to the apical membrane of enterocytes, and we hypothesized that MYO5B may also be responsible for the localization of IMAC proteins. To address this question, we used two different mouse models: 1) neonatal germline MYO5B knockout (MYO5B KO) mice and 2) adult intestinal-specific tamoxifen-inducible VillinCreERT2;MYO5Bflox/flox mice. In control mice, immunostaining revealed that CDHR2, CDHR5, USH1C, and MYO7B were highly enriched at the tips of the microvilli. In contrast, neonatal germline and adult MYO5B-deficient mice showed loss of apical CDHR2, CDHR5, and MYO7B in the brush border and accumulation in a subapical compartment. Colocalization analysis revealed decreased Mander's coefficients in adult inducible MYO5B-deficient mice compared with control mice for CDHR2, CDHR5, USH1C, and MYO7B. Scanning electron microscopy images further demonstrated aberrant microvilli packing in adult inducible MYO5B-deficient mouse small intestine. These data indicate that MYO5B is responsible for the delivery of IMAC components to the apical membrane.NEW & NOTEWORTHY The intestinal epithelium absorbs nutrients and water through an elaborate apical membrane of highly organized microvilli. Microvilli organization is regulated by the intermicrovillar adhesion complexes, which create links between neighboring microvilli and control microvilli packing and density. In this study, we report a new trafficking partner of the IMAC, Myosin 5b. Loss of Myosin 5b results in a disorganized brush border and failure of IMAC proteins to reach the distal tips of microvilli.
Assuntos
Enterócitos , Microvilosidades , Miosina Tipo V , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Enterócitos/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Microvilosidades/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismoRESUMO
Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic ß-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.
Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fusão de Membrana/fisiologia , Modelos Biológicos , Animais , Ligação Competitiva , Cálcio/metabolismo , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Células Cromafins/citologia , Dinaminas/metabolismo , Células Secretoras de Insulina/citologia , Microscopia Confocal , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2's role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Ciliadas Auditivas/metabolismo , Audição , Proteínas com Domínio LIM/metabolismo , Proteínas Nucleares/metabolismo , Estereocílios/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Células Ciliadas Auditivas/fisiologia , Perda Auditiva/genética , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Estereocílios/ultraestruturaRESUMO
Background & Aims: All tissues consist of a distinct set of cell types, which collectively support organ function and homeostasis. Tuft cells are a rare epithelial cell type found in diverse epithelia, where they play important roles in sensing antigens and stimulating downstream immune responses. Exhibiting a unique polarized morphology, tuft cells are defined by an array of giant actin filament bundles that support â¼2 µm of apical membrane protrusion and extend over 7 µm towards the cell's perinuclear region. Despite their established roles in maintaining intestinal epithelial homeostasis, tuft cells remain understudied due to their rarity (e.g. â¼ 1% in the small intestinal epithelium). Details regarding the ultrastructural organization of the tuft cell cytoskeleton, the molecular components involved in building the array of giant actin bundles, and how these cytoskeletal structures support tuft cell biology remain unclear. Methods: To begin to answer these questions, we used advanced light and electron microscopy to perform quantitative morphometry of the small intestinal tuft cell cytoskeleton. Results: We found that tuft cell core bundles consist of actin filaments that are crosslinked in a parallel "barbed-end out" configuration. These polarized structures are also supported by a unique group of tuft cell enriched actin-binding proteins that are differentially localized along the giant core bundles. Furthermore, we found that tuft cell actin bundles are co-aligned with a highly ordered network of microtubules. Conclusions: Tuft cells assemble a cytoskeletal superstructure that is well positioned to serve as a track for subcellular transport along the apical-basolateral axis and in turn, support the dynamic sensing functions that are critical for intestinal epithelial homeostasis. SYNOPSIS: This research leveraged advanced light and electron microscopy to perform quantitative morphometry of the intestinal tuft cell cytoskeleton. Three-dimensional reconstructions of segmented image data revealed a co-aligned actin-microtubule superstructure that may play a fundamental role in tuft cell function.
RESUMO
Bacillus anthracis, a Gram-positive facultative anaerobe and the causative agent of anthrax, multiplies to extraordinarily high numbers in vertebrate blood, resulting in considerable heme exposure. Heme is an essential nutrient and the preferred iron source for bacteria during vertebrate colonization, but its high redox potential makes it toxic in excess. To regulate heme homeostasis, many Gram-positive bacteria, including B. anthracis, rely on the two-component signaling system HssRS. HssRS comprises the heme sensing histidine kinase HssS, which modulates the activity of the HssR transcription factor to enable bacteria to circumvent heme toxicity. However, the regulation of the HssRS system remains unclear. Here we identify FapR, the transcriptional regulator of fatty acid biosynthesis, as a key factor in HssRS function. FapR plays an important role in maintaining membrane integrity and the localization of the histidine kinase HssS. Specifically, disruption of fapR leads to increased membrane rigidity, which hinders the penetration of HssRS inducers, resulting in the inactivation of HssRS. Furthermore, deletion of fapR affects the loading of HssS onto the cell membrane, compromising its heme sensing function and subsequently reducing endogenous heme biosynthesis. These findings shed light on the molecular mechanisms governing bacterial adaptation to heme stress and provide potential targets for antimicrobial intervention strategies.
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Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2α signaling. Identifying means to regulate HIF-2α thus has potential therapeutic benefit. Acetyl-CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2α and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2α levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2α. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF-2α expression and stability. While HIF-2α protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHL-independent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2α and that overexpression of MUL1 decreased HIF-2α levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2α stability and ACSS2 inhibition as a strategy to complement HIF-2α-targeted therapies and deplete pathogenically stabilized HIF-2α.
Assuntos
Acetato-CoA Ligase , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carcinoma de Células Renais , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais , Transdução de Sinais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/genética , Linhagem Celular Tumoral , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/genética , Animais , Camundongos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genéticaRESUMO
Introduction: Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. Methods: We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. Results: There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Discussion: Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.
Assuntos
Dieta Ocidental , Células Secretoras de Insulina , Fenômenos Fisiológicos da Nutrição Materna , Mitocôndrias , Efeitos Tardios da Exposição Pré-Natal , Animais , Feminino , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Gravidez , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dieta Ocidental/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Masculino , LactaçãoRESUMO
Nanoparticles composed of a magnetic iron oxide core surrounded by a metal shell have utility in a broad range of biomedical applications. However, the presence of surface energy differences between the two components makes wetting of oxide with metal unfavorable, precluding a "core-shell" structure of an oxide core completely surrounded by a thin metal shell. Three-dimensional island growth followed by island coalescence into thick shells is favored over the two-dimensional layer-by-layer growth of a thin, continuous metal coating of a true core-shell. Aqueous synthesis of gold-coated magnetite nanoparticles with analysis by infrared, energy-dispersive X-ray, and electron energy loss spectroscopies; high-resolution transmission electron microscopy; selected area electron diffraction; and high-angle annular dark-field scanning transmission electron microscopy showed two distinct morphologies that are inconsistent with an idealized core-shell. The majority were isolated ~16-22-nm-diameter nanoparticles consisting of ~7-nm-diameter magnetite and a thick deposition of gold, most often discontinuous, with some potentially "sandwiched" morphologies. A minority were aggregates of agglomerated magnetite decorated with gold but displaying significant bare magnetite. Both populations were successfully conjugated to fibrinogen and targeted to surface-activated platelets, demonstrating that iron oxide-gold nanoparticles produced by aqueous synthesis do not require an ideal core-shell structure for biological activity in cell labeling and targeting applications.
Assuntos
Plaquetas/metabolismo , Óxido Ferroso-Férrico/metabolismo , Fibrinogênio/metabolismo , Ouro/metabolismo , Nanopartículas/metabolismo , Óxido Ferroso-Férrico/química , Fibrinogênio/química , Ouro/química , Humanos , Nanopartículas/química , Ligação ProteicaRESUMO
Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.
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Differentiated transporting epithelial cells present an extensive apical array of microvilli - a "brush border" - where neighboring microvilli are linked together by intermicrovillar adhesion complexes (IMACs) composed of protocadherins CDHR2 and CDHR5. Although loss-of-function studies provide strong evidence that IMAC function is needed to build a mature brush border, how the IMAC contributes to the stabilization and accumulation of nascent microvilli remains unclear. We found that, early in differentiation, the apical surface exhibits a marginal accumulation of microvilli, characterized by higher packing density relative to medial regions of the surface. While medial microvilli are highly dynamic and sample multiple orientations over time, marginal protrusions exhibit constrained motion and maintain a vertical orientation. Unexpectedly, we found that marginal microvilli span the junctional space and contact protrusions on neighboring cells, mediated by complexes of CDHR2/CDHR5. FRAP analysis indicated that these transjunctional IMACs are highly stable relative to adhesion complexes between medial microvilli, which explains the restricted motion of protrusions in the marginal zone. Finally, long-term live imaging revealed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions of the cell. Collectively, our findings suggest that nascent microvilli are stabilized by a capture mechanism that is localized to cell margins and enabled by the transjunctional formation of IMACs. These results inform our understanding of how apical specializations are assembled in diverse epithelial systems.
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This review provides an overview of the current methods for quantifying mitochondrial ultrastructure, including cristae morphology, mitochondrial contact sites, and recycling machinery and a guide to utilizing electron microscopy to effectively measure these organelles. Quantitative analysis of mitochondrial ultrastructure is essential for understanding mitochondrial biology and developing therapeutic strategies for mitochondrial-related diseases. Techniques such as transmission electron microscopy (TEM) and serial block face-scanning electron microscopy, as well as how they can be combined with other techniques including confocal microscopy, super-resolution microscopy, and correlative light and electron microscopy are discussed. Beyond their limitations and challenges, we also offer specific magnifications that may be best suited for TEM analysis of mitochondrial, endoplasmic reticulum, and recycling machinery. Finally, perspectives on future quantification methods are offered.
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Retículo Endoplasmático , Mitocôndrias , Microscopia Eletrônica de Varredura , Mitocôndrias/ultraestrutura , Microscopia Eletrônica de TransmissãoRESUMO
To maximize solute transport, epithelial cells build an apical "brush border," where thousands of microvilli are linked to their neighbors by protocadherin-containing intermicrovillar adhesion complexes (IMACs). Previous studies established that the IMAC is needed to build a mature brush border, but how this complex contributes to the accumulation of new microvilli during differentiation remains unclear. We found that early in differentiation, mouse, human, and porcine epithelial cells exhibit a marginal accumulation of microvilli, which span junctions and interact with protrusions on neighboring cells using IMAC protocadherins. These transjunctional IMACs are highly stable and reinforced by tension across junctions. Finally, long-term live imaging showed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions. Thus, nascent microvilli are stabilized by a marginal capture mechanism that depends on the formation of transjunctional IMACs. These results may offer insights into how apical specializations are assembled in diverse epithelial systems.