RESUMO
Tissue-resident memory CD8+ T cells (TRM) reside at sites of previous infection, providing protection against reinfection with the same pathogen. In the skin, TRM patrol the epidermis, where keratinocytes are the entry site for many viral infections. Epidermal TRM react rapidly to cognate antigen encounter with the secretion of cytokines and differentiation into cytotoxic effector cells, constituting a first line of defense against skin reinfection. Despite the important protective role of skin TRM, it has remained unclear, whether their reactivation requires a professional antigen-presenting cell (APC). We show here, using a model system that allows antigen targeting selectively to keratinocytes in a defined area of the skin, that limited antigen expression by keratinocytes results in rapid, antigen-specific reactivation of skin TRM. Our data identify epidermal Langerhans cells that cross-present keratinocyte-derived antigens, as the professional APC indispensable for the early reactivation of TRM in the epidermal layer of the skin.
Assuntos
Linfócitos T CD8-Positivos , Células de Langerhans , Humanos , Células T de Memória , Reinfecção/metabolismo , Epiderme , Antígenos , Memória ImunológicaRESUMO
Tert-butyl hydroperoxide (t-BuOOH) is an organic hydroperoxide widely used as a model compound to induce oxidative stress. It leads to a plethora of cellular damage, including lipid peroxidation, DNA double-strand breaks (DNA DSBs), and breakdown of the mitochondrial membrane potential (MMP). We could show in several cell lines that t-BuOOH induces ferroptosis, triggered by iron-dependent lipid peroxidation. We have further revealed that not only t-BuOOH-mediated ferroptosis, but also DNA DSBs and loss of MMP are prevented by cell-cell contacts. The underlying mechanisms are not known. Here, we show in murine fibroblasts and a human colon carcinoma cell line that t-BuOOH (50 or 100 µM, resp.) causes an increase in intracellular Ca2+, and that this increase is key to lipid peroxidation and ferroptosis, DNA DSB formation and dissipation of the MMP. We further demonstrate that cell-cell contacts prevent t-BuOOH-mediated raise in intracellular Ca2+. Hence, we provide novel insights into the mechanism of t-BuOOH-triggered cellular damage including ferroptosis and propose a model in which cell-cell contacts control intracellular Ca2+ levels to prevent lipid peroxidation, DNA DSB-formation and loss of MMP. Since Ca2+ is a central player of toxicity in response to oxidative stress and is involved in various cell death pathways, our observations suggest a broad protective function of cell-cell contacts against a variety of exogenous toxicants.
Assuntos
Cálcio , Quebras de DNA de Cadeia Dupla , Ferroptose , Peroxidação de Lipídeos , Potencial da Membrana Mitocondrial , terc-Butil Hidroperóxido , Ferroptose/efeitos dos fármacos , Cálcio/metabolismo , Humanos , terc-Butil Hidroperóxido/toxicidade , Animais , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Linhagem Celular TumoralRESUMO
Single molecule detection and tracking provides at times the only possible method to observe the interactions of low numbers of biomolecules, inlcuding DNA, receptors and signal mediating proteins in living systems. However, most existing imaging methods do not enable both high sensitivity and non-invasive imaging of large specimens. In this study we report a new setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with the resolution of confocal microscopy and the optical penetration beyond 300 µm. We detect and report our instrumental figures of merit, control values of fluorescence properties of single nano crystals in comparison to both standard widefield configurations, and also values of nanocrystals in multicellular "fruiting bodies" of Dictyostelium, an excellent control as a model developmental system. In the Dictyostelium , we also report some of our first tracking of single nanocrystals with SPIM. The new SPIM setup represents a new technique, which enables fast single molecule imaging and tracking in living systems.
RESUMO
Single molecule detection and tracking provides at times the only possible method to observe the interactions of low numbers of biomolecules, inlcuding DNA, receptors and signal mediating proteins in living systems. However, most existing imaging methods do not enable both high sensitivity and non-invasive imaging of large specimens. In this study we report a new setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with the resolution of confocal microscopy and the optical penetration beyond 300 µm. We detect and report our instrumental figures of merit, control values of fluorescence properties of single nano crystals in comparison to both standard widefield configurations, and also values of nanocrystals in multicellular "fruiting bodies" of Dictyostelium, an excellent control as a model developmental system. In the Dictyostelium , we also report some of our first tracking of single nanocrystals with SPIM. The new SPIM setup represents a new technique, which enables fast single molecule imaging and tracking in living systems.
RESUMO
The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of <100nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca(2+). Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from <0.01µm(2)s(-1) to ~5µm(2) s(-1), whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of >10µm(2)s(-1). For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells.
Assuntos
Calmodulina/análise , Imagem Individual de Molécula , Transporte Biológico , Sinalização do Cálcio , Carbocianinas , Difusão , Ácido Egtázico/análogos & derivados , Recuperação de Fluorescência Após Fotodegradação , Corantes Fluorescentes , Células HEK293 , Humanos , Microinjeções , Ligação Proteica , Frações Subcelulares/químicaRESUMO
Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.
Assuntos
Movimento Celular/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Apoptose , Células HeLa , Humanos , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Peixe-ZebraRESUMO
The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos , Animais , Ânions/metabolismo , Detergentes/farmacologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Espectrometria de Massas , Microscopia Confocal , Microscopia de Fluorescência , Oócitos/citologia , Oócitos/metabolismo , Estrutura Terciária de Proteína , RNA Complementar/metabolismo , Transdução de Sinais , Esteróis/metabolismo , Xenopus/metabolismoRESUMO
VE-cadherin is the predominant adhesion molecule in vascular endothelial cells being responsible for maintenance of the endothelial barrier function by forming adhesive contacts (adherens junctions) to neighbouring cells. We found by use of single molecule fluorescence microscopy that VE-cadherin is localised in preformed clusters when not inside adherens junctions. These clusters depend on the integrity of the actin cytoskeleton and are localised in cholesterol rich microdomains of mature endothelial cells as found by membrane fractionation. The ability to form and maintain VE-cadherin based junctions was probed using the laser tweezer technique, and we found that cholesterol depletion has dramatical effects on VE-cadherin mediated adhesion. While a 30% reduction of the cholesterol-level results in an increase of adhesion, excessive cholesterol depletion by about 60% leads to an almost complete loss of VE-cadherin function. Nevertheless, the cadherin concentration in the membrane and the single molecule kinetic parameters of the cadherin are not changed. Our results suggest that the actin cytoskeleton, junction-associated proteins and protein-lipid assemblies in cholesterol-rich micro-domains mutually stabilise each other to form functional adhesion contacts.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Colesterol/deficiência , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Junções Aderentes/metabolismo , Animais , Western Blotting , Células CHO , Adesão Celular , Cricetinae , Cricetulus , Cães , Imunofluorescência , Cinética , Células Madin Darby de Rim Canino , Microscopia de Vídeo , Pinças Ópticas , Ligação Proteica , Transporte ProteicoRESUMO
Bone (or body) morphogenetic proteins (BMPs) belong to the TGFß superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Membrana Celular/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Membrana Celular/genética , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Transporte Proteico/fisiologia , Proteínas Smad/genéticaRESUMO
Primary cilia are microtubule-based cell organelles important for cellular communication. Since they are involved in the regulation of numerous signalling pathways, defects in cilia development or function are associated with genetic disorders, collectively called ciliopathies. Besides their ciliary functions, recent research has shown that several ciliary proteins are involved in the coordination of the actin cytoskeleton. Although ciliary and actin phenotypes are related, the exact nature of their interconnection remains incompletely understood. Here, we show that the protein BBS6, associated with the ciliopathy Bardet-Biedl syndrome, cooperates with the actin-bundling protein Fascin-1 in regulating filopodia and ciliary signalling. We found that loss of Bbs6 affects filopodia length potentially via attenuated interaction with Fascin-1. Conversely, loss of Fascin-1 leads to a ciliary phenotype, subsequently affecting ciliary Wnt signalling, possibly in collaboration with BBS6. Our data shed light on how ciliary proteins are involved in actin regulations and provide new insight into the involvement of the actin regulator Fascin-1 in ciliogenesis and cilia-associated signalling. Advancing our knowledge of the complex regulations between primary cilia and actin dynamics is important to understand the pathogenic consequences of ciliopathies.
Assuntos
Actinas , Ciliopatias , Humanos , Actinas/metabolismo , Via de Sinalização WntRESUMO
The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding-proteins and nucleation-promoting factors to perform fundamental cellular functions. We have previously shown that ERK3, an atypical MAPK, controls IL-8 production and chemotaxis (Bogueka et al., 2020). Here, we show in human cells that ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent RAC1 and CDC42 activation, maintenance of F-actin content, filopodia formation, and epithelial cell migration. Further, ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. ERK3 kinase activity was required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.
Assuntos
Actinas , Proteína Quinase 6 Ativada por Mitógeno , Animais , Humanos , Actinas/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Polimerização , Movimento Celular , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Mamíferos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Hard tissue formation patterns and rates reveal details of animal physiology, life history, and environment, but are understudied in reptiles. Here, we use fluorescence labels delivered in vivo and laser confocal scanning microscopy to study tooth and bone formation in a managed group of green iguanas (Iguana iguana, Linné 1758) kept for 1.5 years under experimentally controlled conditions and undergoing several dietary switches. We constrain rates of tooth elongation, which we observe to be slow when enamel is initially deposited (c. 9 µm/day), but then increases exponentially in the dentin root, reaching c. 55 µm/day or more after crown completion. We further constrain the total timing of tooth formation to â¼40-60 days, and observe highly variable timings of tooth resorption onset and replacement. Fluorescent labels clearly indicate cohorts of teeth recruited within Zahnreihen replacement waves, with faster sequential tooth recruitment and greater wave sizes posteriorly, where each wave initiates. Fluorescence further reveals enamel maturation after initial deposition. Rates of hard tissue formation in long bones range from 0.4 to 3.4 µm/day, correlating with animal weight gain and cortical bone recording the entire history of the experiment. We suggest additional labeling experiments to study hard tissue formation patterns in other reptiles, and propose strategies for chemical analyses of hard tissues in order to extract temporal information about past environments, behaviors, and diets from reptilian fossils throughout the Phanerozoic.
Assuntos
Iguanas , Dente , Animais , Fluorescência , Osso e Ossos , DietaRESUMO
Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-ß1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.
Assuntos
Insuficiência Cardíaca , Células Mieloides , Infarto do Miocárdio , Animais , Humanos , Camundongos , Fibrose , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Mieloides/metabolismo , Infarto do Miocárdio/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Remodelação VentricularRESUMO
Evidence exists that cAMP stabilizes the endothelial barrier, in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties, similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (EPac/Rap 1) signaling enhanced transendothelial electrical resistance and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin 5. Ultrastructural analysis of the intercellular contact zone architecture documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contact sites displaying complex interdigitations of cell borders, in which membranes of neighboring cells were closely apposed over comparatively long distances; in addition, they were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP, whereas F/R-mediated Rac1 activation and barrier enhancement were not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement, and contact zone reorganization. These results indicate that, in microvascular endothelium, Rac1-mediated alterations in contact zone architecture contribute to cAMP-induced barrier stabilization.
Assuntos
AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Permeabilidade Capilar/fisiologia , Feminino , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Microvasos/metabolismo , Microvasos/ultraestrutura , Ratos , Transdução de Sinais/fisiologiaRESUMO
Microglia are phagocytosis-competent CNS cells comprising a spectrum of subtypes with beneficial and/or detrimental functions in acute and chronic neurodegenerative disorders. The heterogeneity of microglia suggests differences in phagocytic activity and phenotype plasticity between microglia subtypes. To study these issues, primary murine glial cultures were cultivated in the presence of serum, different growth factors and cytokines to obtain M0-like, M1-like, and M2-like microglia as confirmed by morphology, M1/M2 gene marker expression, and nitric oxide assay. Single-cell analysis after 3 hours of phagocytosis of E.coli particles or IgG-opsonized beads showed equal internalization by M0-like microglia, whereas M1-like microglia preferably internalized E.coli particles and M2-like microglia preferably internalized IgG beads, suggesting subtype-specific preferences for different phagocytosis substrates. Time-lapse live-cells imaging over 16 hours revealed further differences between microglia subtypes in phagocytosis preference and internalization dynamics. M0- and, more efficiently, M1-like microglia continuously internalized E.coli particles for 16 hours, whereas M2-like microglia discontinued internalization after approximately 8 hours. IgG beads were continuously internalized by M0- and M1-like microglia but strikingly less by M2-like microglia. M2-like microglia initially showed continuous internalization similar to M0-like microglia but again discontinuation of internalization after 8 hours suggesting that the time of substrate exposure differently affect microglia subtypes. After prolonged exposure to E.coli particles or IgG beads for 5 days all microglia subtypes showed increased internalization of E.coli particles compared to IgG beads, increased nitric oxide release and up-regulation of M1 gene markers, irrespectively of the phagocytosis substrate, suggesting phenotype plasticity. In summary, microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity. The results suggest that prolonged phagocytosis substrate exposure enhances M1-like profiles and M2-M1 repolarization of microglia. Similar processes may also take place in conditions of acute and chronic brain insults when microglia encounter different types of phagocytic substrates.
Assuntos
Microglia , Óxido Nítrico , Animais , Imunoglobulina G/metabolismo , Camundongos , Microglia/metabolismo , Óxido Nítrico/metabolismo , Fagocitose , FenótipoRESUMO
BACKGROUND: Stimulated dendritic cells (DCs), which constitute the most potent population of antigen-presenting cells (APCs), express the actin-bundling protein Fascin-1 (Fscn1). In tumor cells, de novo expression of Fscn1 correlates with their invasive and metastatic properties. Therefore, Fscn1 inhibitors have been developed to serve as antitumor agents. In this study, we were interested in better understanding the impact of Fscn1 inhibitors on DCs. METHODS: In parallel settings, murine spleen cells and bone-marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide in the presence of Fscn1 inhibitors (NP-G2-044 and BDP-13176). An analysis of surface expression of costimulatory and coinhibitory receptors, as well as cytokine production, was performed by flow cytometry. Cytoskeletal alterations were assessed by confocal microscopy. The effects on the interactions of BMDCs with antigen-specific T cells were monitored by time lapse microscopy. The T-cell stimulatory and polarizing capacity of BMDCs were measured in proliferation assays and cytokine studies. RESULTS: Administration of Fscn1 inhibitors diminished Fscn1 expression and the formation of dendritic processes by stimulated BMDCs and elevated CD273 (PD-L2) expression. Fscn1 inhibition attenuated the interaction of DCs with antigen-specific T cells and concomitant T-cell proliferation. CONCLUSIONS: Systemic administration of Fscn1 inhibitors for tumor therapy may also modulate DC-induced antitumor immune responses.
RESUMO
We demonstrate the first, to our knowledge, integration of stimulated emission depletion (STED) with selective plane illumination microscopy (SPIM). Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. The integrated STED-SPIM method combines the advantages of SPIM with the resolution enhancement of STED, and thus provides a method for fast, high-resolution imaging with >100 µm deep penetration into biological tissue.
Assuntos
Iluminação/métodos , Microscopia/métodos , Animais , Embrião não Mamífero , Fenômenos Ópticos , Peixe-Zebra/embriologiaRESUMO
The fluorescein arsenical hairpin binder (FlAsH) shows much promise to determine the relative orientations of protein regions and structures even in living cells and in the plasma membrane. In this study, we characterized FlAsH's photophysical properties by steady-state anisotropy and time-resolved single photon counting for further applications with G-protein coupled receptors. We find that FlAsH has a relatively high initial anisotropy of 0.31 ± 0.01 and a three-component fluorescence lifetime with an average of 4.1 ± 0.1 ns. We characterized the FlAsH fluorophore orientation in the α(2A) adrenergic receptor revealing rigid orientations of FlAsH in the membrane plane for rotational correlation times of â¼50 ns in living cells. To elucidate the fluorophore-membrane orientation and rotational correlation time, an anisotropy treatment similar to that of another researcher (Axelrod, D. 1979. Biophys. J. 26:557-573) was developed. The rotational correlation times were observed to increase by up to 16 ns after agonist addition. The rotational correlation time also allowed for a comparison to the theoretical relationship between translational and rotational diffusion (originally proposed by Saffman, P. G., and M. Delbrück. 1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113) and revealed a discrepancy of a factor between 10 and 100.
Assuntos
Arsenicais/metabolismo , Difusão , Fluoresceína/metabolismo , Fluoresceínas/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Rotação , Coloração e Rotulagem , Animais , Anisotropia , Membrana Celular/metabolismo , Sobrevivência Celular , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Camundongos , Modelos Moleculares , Fatores de TempoRESUMO
Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.
Assuntos
Transporte Axonal/fisiologia , Neurônios Motores/metabolismo , Doenças Priônicas/metabolismo , Amidinas/metabolismo , Animais , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Córtex Motor/metabolismo , Córtex Motor/patologia , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/patologia , Núcleo Rubro/metabolismo , Núcleo Rubro/fisiopatologia , Nervo Isquiático/metabolismo , Nervo Isquiático/ultraestrutura , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The Department of Veterans Affairs (VA's) mission is to care for those who have borne the battle. As medical technology has advanced, more and more of our returning combat Veterans survive what would have been fatal wounds in previous conflicts ( Gawande, 2004 ). But survival is only the immediate goal-our job is to restore Veterans to the greatest level of health, independence, and quality of life that is medically possible. The VA is achieving this goal through close collaboration with the Department of Defense (DoD) to facilitate a smooth transition and continuum of care that ensures Veterans and Service Members receive the care they deserve. This article describes VA's system of Veteran-centered, post-combat care programs that rely on significant involvement of social workers to support Service Members, Veterans and their families through recovery, rehabilitation, and re-integration into their home communities.