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1.
bioRxiv ; 2023 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-37808646

RESUMO

Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.

2.
J Vis Exp ; (141)2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30582601

RESUMO

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (Aß) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). Aß, which forms neurotoxic amyloid aggregates in Alzheimer's disease (AD), has been shown to interact with fibrinogen. This Aß-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. Aß-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the Aß-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of Aß and Aß-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of Aß-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate Aß-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified Aß-fibrinogen interaction inhibitor.


Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Fibrina/metabolismo , Microscopia Eletrônica de Varredura/métodos , Análise Espectral/métodos , Fibrina/análise , Humanos
3.
Proc Natl Acad Sci U S A ; 114(43): E9066-E9075, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29073103

RESUMO

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , DNA Mitocondrial/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Feminino , Fibroblastos/patologia , Transferência Genética Horizontal , Genoma Mitocondrial/genética , Humanos , Células MCF-7 , NADH Desidrogenase/genética , Fosforilação Oxidativa , Receptores de Estrogênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Dev Cell ; 39(4): 424-437, 2016 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-27818179

RESUMO

Vertebrate cells can initiate ciliogenesis from centrioles at the cell center, near the Golgi, forming primary cilia confined or submerged in a deep narrow pit created by membrane invagination. How or why cells maintain submerged cilia is unclear. Here, by characterizing centriole subdistal appendages (sDAP) in cells exclusively growing submerged cilia, we found that a group of sDAP components localize to the centriole proximal end through the cohesion factor C-Nap1 and that sDAP function redundantly with C-Nap1 for submerged cilia maintenance. Loss of sDAP and C-Nap1 has no effect on cilia assembly, but it disrupts stable Golgi-cilia association and allows normally submerged cilia to fully surface, losing the deep membrane invagination. Intriguingly, unlike submerged cilia (stationary), surfaced cilia actively respond to mechanical stimuli with motions and can ectopically recruit Hedgehog signaling components in the absence of agonist. We propose that spatial control of ciliogenesis uncouples or specifies sensory properties of cilia.


Assuntos
Cílios/metabolismo , Morfogênese , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Centríolos/metabolismo , Centríolos/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cílios/ultraestrutura , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas Hedgehog/metabolismo , Humanos , Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Movimento (Física) , Mutação/genética , Reologia , Sensação , Transdução de Sinais
5.
R Soc Open Sci ; 2(11): 150437, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26716000

RESUMO

Microbes living in stagnant water typically rely on chemical diffusion to draw nutrients from their environment. The sulfur-oxidizing bacterium Thiovulum majus and the ciliate Uronemella have independently evolved the ability to form a 'veil', a centimetre-scale mucous sheet on which cells organize to produce a macroscopic flow. This flow pulls nutrients through the community an order of magnitude faster than diffusion. To understand how natural selection led these microbes to evolve this collective behaviour, we connect the physical limitations acting on individual cells to the cell traits. We show how diffusion limitation and viscous dissipation have led individual T. majus and Uronemella cells to display two similar characteristics. Both of these cells exert a force of approximately 40 pN on the water and attach to boundaries by means of a mucous stalk. We show how the diffusion coefficient of oxygen in water and the viscosity of water define the force the cells must exert. We then show how the hydrodynamics of filter-feeding orient a microbe normal to the surface to which it attaches. Finally, we combine these results with new observations of veil formation and a review of veil dynamics to compare the collective dynamics of these microbes. We conclude that this convergent evolution is a reflection of similar physical limitations imposed by diffusion and viscosity acting on individual cells.

6.
Nature ; 527(7578): 329-35, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26524530

RESUMO

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.


Assuntos
Encéfalo/metabolismo , Exossomos/metabolismo , Integrinas/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Tropismo , Animais , Biomarcadores/metabolismo , Encéfalo/citologia , Linhagem Celular Tumoral , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes src , Humanos , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/antagonistas & inibidores , Integrina alfa6beta4/metabolismo , Cadeias beta de Integrinas/metabolismo , Integrina beta4/metabolismo , Integrinas/antagonistas & inibidores , Células de Kupffer/citologia , Células de Kupffer/metabolismo , Fígado/citologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fosforilação , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/metabolismo , Proteínas S100/genética
7.
Hypertension ; 66(1): 175-82, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25941345

RESUMO

Numerous epidemiological studies link vascular disorders, such as hypertension, diabetes mellitus, and stroke, with Alzheimer's disease (AD). Hypertension, specifically, is an important modifiable risk factor for late-onset AD. To examine the link between midlife hypertension and the onset of AD later in life, we chemically induced chronic hypertension in the TgSwDI mouse model of AD in early adulthood. Hypertension accelerated cognitive deficits in the Barnes maze test (P<0.05 after 3 months of treatment; P<0.001 after 6 months), microvascular deposition of ß-amyloid (P<0.001 after 3 months of treatment; P<0.05 after 6 months), vascular inflammation (P<0.05 in the dentate gyrus and P<0.001 in the dorsal subiculum after 6 months of treatment), blood-brain barrier leakage (P<0.05 after 3 and 6 months of treatment), and pericyte loss (P<0.05 in the dentate gyrus and P<0.01 in the dorsal subiculum after 6 months of treatment) in these mice. In addition, hypertension induced hippocampal neurodegeneration at an early age in this mouse line (43% reduction in the dorsal subiculum; P<0.05), establishing this as a useful research model of AD with mixed vascular and amyloid pathologies.


Assuntos
Doença de Alzheimer , Hipocampo/patologia , Hipertensão/complicações , Degeneração Neural/etiologia , Idade de Início , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/análise , Precursor de Proteína beta-Amiloide/genética , Animais , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Angiopatia Amiloide Cerebral/etiologia , Angiopatia Amiloide Cerebral/patologia , Doença Crônica , Demência Vascular/etiologia , Demência Vascular/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microvasos/química , Microvasos/patologia , Mutação , NG-Nitroarginina Metil Éster/toxicidade , Degeneração Neural/fisiopatologia , Pericitos/patologia , Vasculite/etiologia , Vasculite/fisiopatologia
8.
Dev Dyn ; 241(4): 759-69, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22334475

RESUMO

INTRODUCTION: In this study we examined Roundabout signaling in the Drosophila embryonic hindgut. RESULTS: Slit and its receptors Roundabout (Robo) and Roundabout 2 (Robo2) localize to discrete regions in the hindgut epithelium and surrounding visceral mesoderm. Loss of robo, robo2 or slit did not disrupt overall hindgut patterning. However, slit and robo mutants showed a decrease in microvillus length on the boundary cells of the hindgut epithelium. Rescue and overexpression analysis revealed that robo is specifically required in the visceral mesoderm for correct microvillus length in the underlying hindgut epithelium. Expression of robo in the visceral mesoderm of robo mutant embryos restored normal microvillus length, while overexpression of robo resulted in an increase in microvillus length. Microvillus length was also increased in robo2 mutants suggesting that robo2 may antagonize robo function in the hindgut. CONCLUSION: Together, these results establish a novel, dose-dependent role for Robo in regulating microvilli growth and provide in vivo evidence for the role of the visceral mesoderm in controlling morphological changes in the underlying intestinal epithelium.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Embrião não Mamífero , Epitélio/embriologia , Receptores Imunológicos/fisiologia , Animais , Sistema Digestório/citologia , Sistema Digestório/embriologia , Drosophila/fisiologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/fisiologia , Epitélio/ultraestrutura , Mesoderma/embriologia , Mesoderma/fisiologia , Microvilosidades/ultraestrutura , Transdução de Sinais
9.
J Vis Exp ; (34)2009 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-20027180

RESUMO

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.


Assuntos
Drosophila/embriologia , Drosophila/ultraestrutura , Coração/embriologia , Microscopia Eletrônica/métodos , Miocárdio/ultraestrutura , Animais
10.
J Cell Biol ; 182(2): 241-8, 2008 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-18663139

RESUMO

During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad-mediated cell adhesion.


Assuntos
Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Coração/embriologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Caderinas/genética , Adesão Celular/genética , Diferenciação Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Polaridade Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/fisiopatologia , Neovascularização Fisiológica/genética , Proteínas do Tecido Nervoso/genética , Organogênese/genética , Receptores Imunológicos/genética , Proteínas Roundabout
11.
Proc Natl Acad Sci U S A ; 103(33): 12441-6, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16888037

RESUMO

Heart morphogenesis requires the coordinated regulation of cell movements and cell-cell interactions between distinct populations of cardiac precursor cells. Little is known about the mechanisms that organize cardiac cells into this complex structure. In this study, we analyzed the role of Slit, an extracellular matrix protein and its transmembrane receptors Roundabout (Robo) and Roundabout2 (Robo2) during morphogenesis of the Drosophila heart tube, a process analogous to early heart formation in vertebrates. During heart assembly, two types of progenitor cells align into rows and coordinately migrate to the dorsal midline of the embryo, where they merge to assemble a linear heart tube. Here we show that cardiac-specific expression of Slit is required to maintain adhesion between cells within each row during dorsal migration. Moreover, differential Robo expression determines the relative distance each row is positioned from the dorsal midline. The innermost CBs express only Robo, whereas the flanking pericardial cells express both receptors. Removal of robo2 causes pericardial cells to shift toward the midline, whereas ectopic robo2 in CBs drives them laterally, resulting in an unfused heart tube. We propose a model in which Slit has a dual role during assembly of the linear heart tube, functioning to regulate both cell positioning and adhesive interactions between migrating cardiac precursor cells.


Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Morfogênese , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Adesão Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Coração/anatomia & histologia , Coração/embriologia , Proteínas do Tecido Nervoso/genética , Fenótipo , Receptores Imunológicos/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Transgenes , Proteínas Roundabout
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