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1.
Nat Commun ; 12(1): 61, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397928

RESUMO

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.


Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Animais , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Lipoilação , Luciferases/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos
2.
Nat Commun ; 12(1): 263, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431828

RESUMO

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. While SVs are mobile within the clusters, the clusters have no boundaries consistent with a liquid phase. We previously found that purified synapsin, a peripheral SV protein, can assemble into liquid condensates and trap liposomes into them. How this finding relates to the physiological formation of SV clusters in living cells remains unclear. Here, we report that synapsin alone, when expressed in fibroblasts, has a diffuse cytosolic distribution. However, when expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. This minimal reconstitution system can be a powerful model to gain mechanistic insight into the assembly of structures which are of fundamental importance in synaptic transmission.


Assuntos
Neurônios/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , Animais , Células COS , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citosol/metabolismo , Endocitose , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Camundongos , Eletricidade Estática , Vesículas Sinápticas/ultraestrutura
3.
Clin Ter ; 171(5): e431-e436, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32901788

RESUMO

AIM OF THE STUDY: To investigate, in vivo and in vitro, the fibroblast-to-myofibroblast transition in patients with hypermobile Ehlers-Danlos Syndrome (EDS). To analyze the dermis of patients with classical form of EDS (cEDS) and with hEDS, to identify qualitative and/or quantitative differences in ECM component and ultrastructural changes in collagen. MATERIALS AND METHODS: Seven subjects, aged over 18, two with cEDS and five with hEDS underwent two skin biopsy. One sample was prepared for transmission electron microscopy (TEM), the other for immunofluorescence. The diameter of collagen fibers was measured with TEM. Fibrils were analyzed in four patients: the two with cEDS and two with hEDS. For each patient, the diameter of n=250 collagen fibrils was measured. αSMA was used as specific marker for myofibroblast to highlight their presence in vivo in the skin of patients with hEDS. RESULT: IF observation could not assess an increased expression of αSMA in hEDS patients, which showed no statistical difference compared to classic form patients. The major result from the analysis of TEM images is the clear difference in ECM composition between the two forms of EDS: ECM in hEDS is optically more dense and more prominently composed of elastic fibers. CONCLUSION: Our study provides the following important evidence: 1) the absence in vivo of dermal fibroblasts in patients with hEDS, demonstrated by αSMA negativity; 2) the presence of statistically significant changes in the diameter of collagen fibrils between the classic and the hypermobile forms.


Assuntos
Síndrome de Ehlers-Danlos/patologia , Fibroblastos/ultraestrutura , Pele/ultraestrutura , Actinas/metabolismo , Adulto , Colágeno/ultraestrutura , Síndrome de Ehlers-Danlos/metabolismo , Humanos
4.
Toxicol Lett ; 333: 105-114, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32736005

RESUMO

Maduramicin frequently induces severe cardiotoxicity in broiler chickens as well as in humans who consume maduramicin accidentally. Apoptosis and non-apoptotic cell death occur concurrently in the process of maduramicin-induced cardiotoxicity; however, the underlying mechanism of non-apoptotic cell death is largely unknown. Here, we report the relationship between maduramicin-caused cytoplasmic vacuolization and methuosis-like cell death as well as the underlying mechanism in primary chicken myocardial cells. Maduramicin induced a significant increase of cytoplasmic vacuoles with a degree of cell specificity in primary chicken embryo fibroblasts and chicken hepatoma cells (LMH), along with a decrease of ATP and an increase of LDH. The accumulated vacuoles were partly derived from cellular endocytosis rather than the swelling of endoplasm reticulum, lysosomes, and mitochondria. Moreover, the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) did not prevent maduramicin-induced cytoplasmic vacuolization. DNA ladder and cleavage of PARP were not observed in chicken myocardial cells during maduramicin exposure. Pretreatment with 3-methyladenine (3-MA) and cholorquine (CQ) of chicken myocardial cells did not attenuate cytoplasmic vacuolization and cytotoxicity, although LC3 and p62 were activated. Bafilomycin A1 almost completely prevented the generation of cytoplasmic vacuoles and significantly attenuated cytotoxicity induced by maduramicin, along with downregulation of K-Ras and upregulation of Rac1. Taken together, "methuosis" due to excessive cytoplasmic vacuolization mediates the cardiotoxicity of maduramicin. This provides new insights for understanding a nonclassical form of cell death in the field of drug-induced cytotoxicity.


Assuntos
Morte Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Lactonas/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Drogas Veterinárias/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Galinhas , Citoplasma , Fragmentação do DNA/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Cultura Primária de Células , Imagem com Lapso de Tempo , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestrutura
5.
Nat Commun ; 11(1): 4102, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796823

RESUMO

Emerging evidence suggests that intestinal stromal cells (IntSCs) play essential roles in maintaining intestinal homeostasis. However, the extent of heterogeneity within the villi stromal compartment and how IntSCs regulate the structure and function of specialized intestinal lymphatic capillary called lacteal remain elusive. Here we show that selective hyperactivation or depletion of YAP/TAZ in PDGFRß+ IntSCs leads to lacteal sprouting or regression with junctional disintegration and impaired dietary fat uptake. Indeed, mechanical or osmotic stress regulates IntSC secretion of VEGF-C mediated by YAP/TAZ. Single-cell RNA sequencing delineated novel subtypes of villi fibroblasts that upregulate Vegfc upon YAP/TAZ activation. These populations of fibroblasts were distributed in proximity to lacteal, suggesting that they constitute a peri-lacteal microenvironment. Our findings demonstrate the heterogeneity of IntSCs and reveal that distinct subsets of villi fibroblasts regulate lacteal integrity through YAP/TAZ-induced VEGF-C secretion, providing new insights into the dynamic regulatory mechanisms behind lymphangiogenesis and lymphatic remodeling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fibroblastos/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Transcrição/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Fibroblastos/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Hibridização in Situ Fluorescente , Mucosa Intestinal/ultraestrutura , Linfangiogênese/genética , Linfangiogênese/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fator C de Crescimento do Endotélio Vascular/genética
6.
Nat Cell Biol ; 22(4): 498-511, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203420

RESUMO

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.


Assuntos
Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Integrinas/genética , Mecanotransdução Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células COS , Adesão Celular , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Biologia Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Pan troglodytes , Domínios Proteicos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/classificação , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
Nat Commun ; 11(1): 519, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980640

RESUMO

Fibroblastic reticular cells (FRCs) are immunologically specialized myofibroblasts of lymphoid organ, and FRC maturation is essential for structural and functional properties of lymph nodes (LNs). Here we show that YAP and TAZ (YAP/TAZ), the final effectors of Hippo signaling, regulate FRC commitment and maturation. Selective depletion of YAP/TAZ in FRCs impairs FRC growth and differentiation and compromises the structural organization of LNs, whereas hyperactivation of YAP/TAZ enhances myofibroblastic characteristics of FRCs and aggravates LN fibrosis. Mechanistically, the interaction between YAP/TAZ and p52 promotes chemokine expression that is required for commitment of FRC lineage prior to lymphotoxin-ß receptor (LTßR) engagement, whereas LTßR activation suppresses YAP/TAZ activity for FRC maturation. Our findings thus present YAP/TAZ as critical regulators of commitment and maturation of FRCs, and hold promise for better understanding of FRC-mediated pathophysiologic processes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Fibroblastos/metabolismo , Linfonodos/citologia , Transativadores/metabolismo , Adipócitos/metabolismo , Animais , Quimiocinas/metabolismo , Fibroblastos/ultraestrutura , Linfonodos/ultraestrutura , Receptor beta de Linfotoxina/metabolismo , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo
8.
Nat Med ; 26(1): 98-109, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932796

RESUMO

Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.


Assuntos
Anormalidades Múltiplas/patologia , Bancos de Espécimes Biológicos , Fatores de Troca do Nucleotídeo Guanina/genética , Fenômica , Proteínas de Peixe-Zebra/genética , Animais , Comportamento Animal , Condrócitos/patologia , Condrócitos/ultraestrutura , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Modelos Biológicos , Sistema Musculoesquelético/patologia , Osteogênese , Fenótipo , Pró-Colágeno/metabolismo , Transporte Proteico , Via Secretória , Síndrome , Peixe-Zebra
9.
Neurology ; 94(5): e474-e480, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31892634

RESUMO

OBJECTIVE: Varicella zoster virus (VZV) can spread anterogradely and infect cerebral arteries causing VZV vasculopathy and arterial ischemic stroke. In this study, we tested the hypothesis that virus-infected cerebrovascular fibroblasts undergo phenotypic changes that promote vascular remodeling and facilitate virus transmission in an in vitro model of VZV vasculopathy. The aims of this project were therefore to examine the changes that virus-infected human brain adventitial vascular fibroblasts (HBVAFs) undergo in an in vitro model of VZV vasculopathy and to identify disease biomarkers relating to VZV-related vasculopathy. METHODS: HBVAFs were infected with VZV, and their ability to migrate, proliferate, transdifferentiate, and interact with endothelial cells was studied with flow cytometry. Microparticles (MPs) released from these cells were isolated and imaged with transmission electron microscopy, and their protein content was analyzed with mass spectrometry. Circulating MP profiles were also studied in children with VZV and non-VZV vasculopathy and compared with controls. RESULTS: VZV-infected HBVAFs transdifferentiated into myofibroblasts with enhanced proliferative and migratory capacity. Interaction of VZV-infected HBVAFs with endothelial cells resulted in endothelial dysfunction. These effects were, in part, mediated by the release of MPs from VZV-infected HBVAFs. These MPs contained VZV virions that could transmit VZV to neighboring cells, highlighting a novel model of VZV cell-to-cell viral dissemination. MPs positive for VZV were significantly higher in children with VZV-related vasculopathy compared to children with non-VZV vasculopathy (p = 0.01) and controls (p = 0.007). CONCLUSIONS: VZV-infected HBVAFs promote vascular remodeling and facilitate virus transmission. These effects were mediated by the release of apoptotic MPs that could transmit VZV infection to neighboring cells through a Trojan horse means of productive viral infection. VZV+ MPs may represent a disease biomarker worthy of further study.


Assuntos
Movimento Celular , Proliferação de Células , Transdiferenciação Celular , Micropartículas Derivadas de Células/virologia , Transtornos Cerebrovasculares/virologia , Fibroblastos/virologia , Miofibroblastos/virologia , Remodelação Vascular , Adolescente , Túnica Adventícia , Micropartículas Derivadas de Células/ultraestrutura , Artérias Cerebrais , Transtornos Cerebrovasculares/fisiopatologia , Criança , Pré-Escolar , Células Endoteliais , Feminino , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Herpesvirus Humano 3 , Humanos , Técnicas In Vitro , Masculino , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Miofibroblastos/fisiologia , Miofibroblastos/ultraestrutura , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/virologia , Cultura de Vírus
10.
Vet Ophthalmol ; 23(1): 113-122, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31328853

RESUMO

This study aimed at examining the histological structure of the pecten oculi in the adult yellow-legged gull, Larus michahellis, and at two moments of postnatal development: during the posthatch (nestling) and juvenile periods. Particular attention was paid to differences in the diameter of vessels, the thickness of the basement membrane, and ultrastructural features of endothelial and pigmented stromal cells. Capillary endothelial cells displayed numerous microvillous-like folds projecting from their internal and external surfaces. Intercellular spaces between capillaries were occupied by pigmented stromal cells. The ultrastructure of pecten oculi underwent noticeable changes during postnatal development. The examination of the capillaries in nestlings, juveniles, and adults revealed that the formation process of vessels and pigmented stromal cells did not complete itself in the posthaching phase. The prominent feature of endothelial cells of capillaries in nestlings was that the microvilli were longer than in juvenile and adult cells, and the capillary lumen was therefore reduced. In this sense, their pigmented stromal cells showed fewer melanosomes, lacked intercellular spaces, and cellular junctions could still be observed. These results provide evidence that the pecten oculi during the posthatching phase maintains immature morphological features consistent with a role of pigmented stromal cells in the blood-retina barrier.


Assuntos
Envelhecimento , Charadriiformes/anatomia & histologia , Vasos Retinianos/ultraestrutura , Animais , Fibroblastos/ultraestrutura , Pericitos/ultraestrutura
11.
Auris Nasus Larynx ; 47(1): 98-104, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31272842

RESUMO

OBJECTIVE: Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) is a disease that features a mechanical dysfunction involving chronic inflammation and altered tissue remodeling. In this study, we aim to evaluate the fibroblast morphology and its cellular traction force in primary fibroblasts cell cultures obtained from both healthy individuals (n=7) and patients with CRSwNP (n=8). METHODS: Using a Traction-force Microscopy we analyzed parameters of Force/Tension in fibroblasts cultures in both experimental groups. RESULTS: The analysis of the Projected Area of Cell revealed that fibroblasts derived from nasal mucosa of healthy individuals have an area on average 39.24% larger than the fibroblasts obtained from the nasal polyp tissue. We also observed that the parameters directly related to the force of the cell, Max Cumulative Force and Net Contractile Moment, presented a high Force/Tension per unit of area in the fibroblasts derived from the healthy nasal mucosa (on average 41% and 52.54% higher than the fibroblasts of the nasal polyp respectively). CONCLUSION: Our results demonstrate a cellular mechanism that may be associated with the mechanical dysfunction found in the Nasal Polyp tissue. The weak traction force of nasal polyp-derived fibroblast may, in lower dimensions, impact on the remodeling of nasal mucosa in CRSwNP.


Assuntos
Fenômenos Biomecânicos , Fibroblastos/ultraestrutura , Pólipos Nasais/ultraestrutura , Pseudópodes/ultraestrutura , Estudos de Casos e Controles , Doença Crônica , Feminino , Fibroblastos/patologia , Fibroblastos/fisiologia , Humanos , Masculino , Microscopia de Força Atômica , Microscopia de Contraste de Fase , Pessoa de Meia-Idade , Pólipos Nasais/patologia , Pólipos Nasais/fisiopatologia , Cultura Primária de Células , Pseudópodes/patologia , Rinite/patologia , Sinusite/patologia
12.
Biochem Biophys Res Commun ; 521(2): 310-317, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31668813

RESUMO

AIM OF THE STUDY: Osteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts. MATERIALS AND METHODS: Fibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled 14C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content. RESULTS AND CONCLUSIONS: Osteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms.


Assuntos
Síndrome de Ehlers-Danlos/patologia , Matriz Extracelular/ultraestrutura , Fibroblastos/patologia , Osteogênese Imperfeita/patologia , Adulto , Anisotropia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Células Cultivadas , Colágeno Tipo I/genética , Feminino , Fibroblastos/ultraestrutura , Glicosaminoglicanos/análise , Humanos , Masculino , Mutação , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética
13.
Spectrochim Acta A Mol Biomol Spectrosc ; 227: 117682, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31672377

RESUMO

The results of the study of composites based on bovine serum albumin (BSA) and single-walled carbon nanotubes (SWCNT) are presented. Nanocomposites were created by evaporation of the water-albumin dispersion with nanotubes using diode laser with temperature control. Two types of nanotubes were used. SWCNT I were synthesized using the electric arc method, SWCNT II were synthesized using the gas phase method. SWCNT I had a diameter and length less than SWCNT II. The mechanism of interaction between BSA and SWCNT in solid nanocomposites is considered. An experimental and theoretical studies of the interaction between aspartic (Asp) and glutamic (Glu) amino acids located on the outer surface of BSA and nanotubes using of vibrational spectroscopy (Fourier-transform infrared (FTIR) and Raman spectroscopy) was carried out. The possibility of nanotubes functionalization by oxygen atoms of negative amino acid residues Asp and Glu, which are on the outer surface of BSA, is shown by molecular modeling. The formation of covalent bonds between BSA and SWCNT in nanocomposites with different concentrations of nanotubes (0.01, 0.1 and 1 g/l) was confirmed by vibrational spectra. The covalent interaction between BSA with SWCNT under the laser irradiation leads to the conformational changes in the secondary and tertiary structures of albumin. This is confirmed by a significant decrease in the intensity of the absorption bands in the high-frequency region. The calculation of the vibrational spectra of the three Glycine:Glycine, Glutamic acid:Threonine and Aspartic acid:Lysine complexes, which take into account hydrogen, ion-dipole and ion-ion bonds, showed that a disturbance in the intermolecular interaction between amino acid residues led to significant decrease in the intensity of absorption bands in the region of stretching vibrations bonds OH and NH. From the Raman spectra, it was found that a significant number of defects in SWCNT is caused by the covalent attachment of oxygen atoms to the graphene surface of nanotubes. An increase in the diameter of nanotubes (4 nm) has practically no effect on the absorption spectrum of nanocomposite, while measuring the concentration of SWCNT affects the FTIR spectra. This confirmed the hydrophobic interaction between BSA and SWCNT. Thus, it was shown that BSA solid nanocomposites with CNTs can interact either with the help of hydrophobic forces or with the formation of covalent bonds, which depends on the diameter of the used nanotubes. The viability of connective fibroblast tissue cells on nanocomposites with both types of SWCNT was demonstrated. It was found that nanocomposites based on SWCNT I provide slightly better compatibility of their structure with fibroblasts. It allows to achieve better cell adhesion to the nanocomposite surface. These criteria make extensive use of scaffold nanocomposites in biomedicine, depending on the requirements for their quality and application.


Assuntos
Nanocompostos/química , Nanotubos de Carbono/química , Soroalbumina Bovina/metabolismo , Análise Espectral Raman , Vibração , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Humanos , Nanotubos de Carbono/ultraestrutura , Tamanho da Partícula , Ligação Proteica , Domínios Proteicos , Teoria Quântica , Soroalbumina Bovina/química , Espectroscopia de Infravermelho com Transformada de Fourier
14.
Brain ; 143(1): 94-111, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31855247

RESUMO

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.


Assuntos
Antígenos CD/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Adolescente , Ataxia/genética , Ataxia/fisiopatologia , Atrofia , Cerebelo/diagnóstico por imagem , Cerebelo/patologia , Colina/farmacologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/fisiopatologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/ultraestrutura , Transtornos de Deglutição/genética , Transtornos de Deglutição/fisiopatologia , Disartria/genética , Disartria/fisiopatologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Incontinência Fecal/genética , Incontinência Fecal/fisiopatologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Mutação da Fase de Leitura , Globo Pálido/diagnóstico por imagem , Transtornos Heredodegenerativos do Sistema Nervoso/diagnóstico por imagem , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/fisiopatologia , Homozigoto , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/genética , Leucoencefalopatias/fisiopatologia , Imagem por Ressonância Magnética , Masculino , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Nootrópicos/farmacologia , Atrofia Óptica/genética , Atrofia Óptica/fisiopatologia , Linhagem , Ribossomos/efeitos dos fármacos , Ribossomos/ultraestrutura , Substância Negra/diagnóstico por imagem , Síndrome , Tremor/genética , Tremor/fisiopatologia , Incontinência Urinária/genética , Incontinência Urinária/fisiopatologia
15.
J Synchrotron Radiat ; 27(Pt 1): 185-198, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31868751

RESUMO

Friedreich's ataxia (FRDA) is a neurodegenerative disease characterized by an increase in intracytoplasmic iron concentration. Here the nanoscale iron distribution within single fibroblasts from FRDA patients was investigated using synchrotron-radiation-based nanoscopic X-ray fluorescence and X-ray in-line holography at the ID16A nano-imaging beamline of the ESRF. This unique probe was deployed to uncover the iron cellular two-dimensional architecture of freeze-dried FRDA fibroblasts. An unsurpassed absolute detection capability of 180 iron atoms within a 30 nm × 50 nm nanoscopic X-ray beam footprint was obtained using state-of-the-art X-ray focusing optics and a large-solid-angle detection system. Various micrometre-sized iron-rich organelles could be revealed for the first time, tentatively identified as endoplasmic reticulum, mitochondria and lysosomes. Also a multitude of nanoscopic iron hot-spots were observed in the cytosol, interpreted as chaperoned iron within the fibroblast's labile iron pool. These observations enable new hypotheses on the storage and trafficking of iron in the cell and ultimately to a better understanding of iron-storage diseases such as Friedreich's ataxia.


Assuntos
Fibroblastos/química , Ataxia de Friedreich/patologia , Holografia/métodos , Ferro/análise , Análise de Célula Única/métodos , Espectrometria por Raios X/métodos , Carbono , Citoplasma/química , Fibroblastos/ultraestrutura , Liofilização , Humanos , Nanoestruturas , Organelas/química , Organelas/ultraestrutura , Análise de Célula Única/instrumentação , Síncrotrons , Fixação de Tecidos/métodos
16.
Hum Mol Genet ; 29(2): 177-188, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31868880

RESUMO

Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5'-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense mutation in the GTPase domain of DRP1 (NM_012062.3:c.436G>A, NP_036192.2: p.D146N variant in DNM1L). Patient's fibroblasts show hyperfused/balloon-like giant mitochondria, highlighting the importance of D146 residue for DRP1 function. This dramatic mitochondrial rearrangement phenocopies what observed overexpressing DRP1-K38A, a well-known experimental dominant negative version of DRP1. In addition, we demonstrated that p.D146N mutation has great impact on peroxisomal shape and function. The p.D146N mutation compromises the GTPase activity without perturbing DRP1 recruitment or assembly, causing decreased mitochondrial and peroxisomal turnover. In conclusion, our findings highlight the importance of sensory neuropathy in the clinical spectrum of DRP1 variants and, for the first time, the impact of DRP1 mutations on mitochondrial turnover and peroxisomal functionality.


Assuntos
Dinaminas/genética , Fibroblastos/ultraestrutura , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Doenças do Sistema Nervoso Periférico/genética , Autofagia/genética , Pré-Escolar , Dinaminas/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Heterozigoto , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação , Linhagem , Doenças do Sistema Nervoso Periférico/enzimologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequenciamento Completo do Exoma
17.
Med Mol Morphol ; 53(1): 7-14, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31104131

RESUMO

We examined the ultrastructure of the anterior cruciate ligament and assessed age-related changes by comparing the ligaments of young and old monkeys. Ultrathin sections of the anterior cruciate ligament were observed by transmission electron microscopy. The three-dimensional architecture of collagen fibers in the ligament was examined by scanning electron microscopy after tissue specimens were treated with 2 N NaOH to digest the extracellular matrix. At the surface layer of the cruciate ligament in young monkeys, fusiform-shaped fibroblasts actively produced collagen fibrils. The ligament consisted of parallel bundles of dense collagen fibrils of approximately 200 nm in diameter. Collagen fibrils appeared to run linearly. Ligament fibrocytes in the deep layer had a stellate form. Ligament fibrocytes decreased in number and showed marked atrophy in old age. Collagen fibrils had a looser configuration in older monkeys. Despite atrophy of fibroblasts in the deep layer of the anterior cruciate ligament, the area with atrophic fibroblasts in the ligament expands with age, which can likely cause deterioration of and a reduction in collagen fibers. This information can be applied in studies on the cause of the low repair ability of and aging-related changes in the anterior cruciate ligament in humans.


Assuntos
Envelhecimento/fisiologia , Ligamento Cruzado Anterior/ultraestrutura , Colágeno/ultraestrutura , Fibroblastos/ultraestrutura , Articulação do Joelho/ultraestrutura , Animais , Macaca fuscata , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microtomia
18.
Cell Rep ; 29(13): 4608-4619.e4, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875564

RESUMO

Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. Palmitoyl-coenzyme A (CoA), the palmitate donor, is produced in the cytosol and is not permeable across membrane bilayers. It is not known how palmitoyl-CoA crosses the ER membrane to access the active site of Hhat. Here, we use fluorescent and radiolabeled palmitoyl-CoA probes to demonstrate that Hhat promotes the uptake of palmitoyl-CoA across the ER membrane in microsomes and semi-intact cells. Reconstitution of purified Hhat into liposomes provided further evidence that palmitoyl-CoA uptake activity is an intrinsic property of Hhat. Palmitoyl-CoA uptake was regulated by and could be uncoupled from Hhat enzymatic activity, implying that Hhat serves a dual function as a palmitoyl acyltransferase and a conduit to supply palmitoyl-CoA to the luminal side of the ER.


Assuntos
Aciltransferases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Hedgehog/metabolismo , Microssomos/metabolismo , Palmitoil Coenzima A/metabolismo , Processamento de Proteína Pós-Traducional , Aciltransferases/genética , Animais , Transporte Biológico , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Lipoilação , Camundongos , Microssomos/ultraestrutura , Transdução de Sinais , Coloração e Rotulagem/métodos
19.
Mutat Res ; 847: 503105, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31699341

RESUMO

18F-FDG PET/CT imaging is used in the diagnosis of diseases, including cancers. The principal photons used for imaging are 511 ke V gamma photons resulting from positron annihilation. The absorbed dose varies among body organs, depending on administered radioactivity and biological clearance. We have attempted to evaluate DNA double-strand breaks (DSB) and toxicity induced in V79 lung fibroblast cells in vitro by 18F-FDG, at doses which might result from PET procedures. Cells were irradiated by 18F-FDG at doses (14.51 and 26.86 mGy), comparable to absorbed doses received by critical organs during PET procedures. The biological endpoints measured were formation of γ-H2AX foci, mitochondrial stress, chromosomal aberrations, and cell cycle perturbation. Irradiation induced DSB (γH2AX assay), mitochondrial depolarization, and both chromosome and chromatid types of aberrations. At higher radiation doses, increased aneuploidy and reduced mitotic activity were also seen. Thus, significant biological effects were observed at the doses delivered by the 18F-FDG exposure and the effects increased with dose.


Assuntos
Aberrações Cromossômicas , Dano ao DNA , Fibroblastos/efeitos da radiação , Radioisótopos de Flúor/toxicidade , Fluordesoxiglucose F18/toxicidade , Raios gama/efeitos adversos , Compostos Radiofarmacêuticos/toxicidade , Aneuploidia , Animais , Benzimidazóis , Carbocianinas , Ciclo Celular/efeitos da radiação , Linhagem Celular , Cromátides/efeitos da radiação , Cromátides/ultraestrutura , Cromossomos/efeitos da radiação , Cromossomos/ultraestrutura , Cricetulus , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Relação Dose-Resposta à Radiação , Fibroblastos/ultraestrutura , Histonas/genética , Cariotipagem , Pulmão/citologia , Masculino , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitose/efeitos da radiação
20.
Cells ; 8(11)2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31766310

RESUMO

Metastatic spread is mainly sustained by cancer stem cells (CSC), a subpopulation of cancer cells that displays stemness features. CSC are thought to be derived from cancer cells that undergo epithelial to mesenchymal transition (EMT), thus acquiring resistance to anoikis and anti-cancer drugs. After detachment from the primary tumor mass, CSC reach the blood and lymphatic flow, and disseminate to the target tissue. This process is by nature dynamic and in vitro models are quite far from the in vivo situation. In this study, we have tried to reproduce the adhesion process of CSC to a target tissue by using a 3D dynamic cell culture system. We isolated two populations of 3D tumor spheroids displaying CSC-like features from breast carcinoma (MCF-7) and lung carcinoma (A549) cell lines. Human fibroblasts were layered on a polystyrene scaffold placed in a dynamically perfused millifluidic system and then the adhesion of tumor cell derived from spheroids to fibroblasts was investigated under continuous perfusion. After 24 h of perfusion, we found that spheroid cells tightly adhered to fibroblasts layered on the scaffold, as assessed by a scanning electron microscope (SEM). To further investigate mechanisms involved in spheroid cell adhesion to fibroblasts, we tested the effect of three RGD integrin antagonists with different molecular structures on cell adhesion; when injected into the circuit, only cilengitide was able to inhibit cell adhesion to fibroblasts. Although our model needs further refinements and improvements, we do believe this study could represent a promising approach in improving current models to study metastatic infiltration in vitro and a new tool to screen new potential anti-metastatic molecules.


Assuntos
Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Esferoides Celulares , Células Tumorais Cultivadas , Biomarcadores , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Expressão Gênica , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/ultraestrutura , Fenótipo , Esferoides Celulares/efeitos dos fármacos
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