RESUMEN
How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin ß1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.
Asunto(s)
Membrana Basal/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Membrana Basal/ultraestructura , Proteínas de Unión al Calcio , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Humanos , Integrinas/metabolismo , Laminina/deficiencia , Laminina/metabolismo , Ratones , Neovascularización Fisiológica , Receptores Notch/antagonistas & inhibidoresRESUMEN
Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofed animals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cell morphology and rearrangement of cytoplasmic membranes, which are a consequence of virus replication. We show here, by confocal immunofluorescence and electron microscopy, that the changes in morphology of FMDV-infected cells involve changes in the distribution of microtubule and intermediate filament components during infection. Despite the continued presence of centrosomes in infected cells, there is a loss of tethering of microtubules to the microtubule organizing center (MTOC) region. Loss of labeling for gamma-tubulin, but not pericentrin, from the MTOC suggests a targeting of gamma-tubulin (or associated proteins) rather than a total breakdown in MTOC structure. The identity of the FMDV protein(s) responsible was determined by the expression of individual viral nonstructural proteins and their precursors in uninfected cells. We report that the only viral nonstructural protein able to reproduce the loss of gamma-tubulin from the MTOC and the loss of integrity of the microtubule system is FMDV 3C(pro). In contrast, infection of cells with another picornavirus, bovine enterovirus, did not affect gamma-tubulin distribution, and the microtubule network remained relatively unaffected.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Citoesqueleto/metabolismo , Enterovirus Bovino/fisiología , Virus de la Fiebre Aftosa/fisiología , Proteínas Virales/metabolismo , Proteasas Virales 3C , Animales , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Tubulina (Proteína)/metabolismoRESUMEN
Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.
Asunto(s)
ADN/administración & dosificación , Integrinas/metabolismo , Lípidos/química , Péptidos/química , Polietilenglicoles/química , Transfección , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Microscopía por Crioelectrón , Endosomas/metabolismo , Humanos , Metabolismo de los Lípidos , Lípidos/síntesis química , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/metabolismo , Plásmidos/administración & dosificación , Polietilenglicoles/síntesis química , Polietilenglicoles/metabolismoRESUMEN
The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti-ICAM-1 and anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Comunicación Celular/inmunología , Células Dendríticas/inmunología , Sinapsis Inmunológicas/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Recuperación de Fluorescencia tras Fotoblanqueo , Molécula 1 de Adhesión Intercelular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Podosomas/metabolismoRESUMEN
Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development.
Asunto(s)
Extensiones de la Superficie Celular/metabolismo , Ectodermo/citología , Ectodermo/enzimología , Cresta Neural/embriología , Tubo Neural/embriología , Neuropéptidos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Ratones , NeurulaciónRESUMEN
Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used in vitro clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.
Asunto(s)
Comunicación Celular , Células Epiteliales/citología , Epitelio Corneal/citología , Limbo de la Córnea/citología , Limbo de la Córnea/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Biomarcadores/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Melanocitos/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Nicho de Células Madre , Células Madre/citología , Células Madre/metabolismo , Células Madre/ultraestructura , Células del Estroma/ultraestructuraRESUMEN
The study of a biological event within a live model organism has become routine through the use of fluorescent labeling of specific proteins in conjunction with laser confocal imaging. These methods allow 3D visualization of temporal events that can elucidate biological function but cannot resolve the tissue organization, extracellular and subcellular details of the tissues. Here, we present a method for correlating electron microscopy image data with the light microscopy data from the same sample volume to reveal the 3D structural information: "correlative light and volume electron microscopy." The methods for live video confocal microscopy, fixation and embedding of the tissue for electron microscopy, the focused ion beam scanning electron microscopy method for sequentially slicing and imaging the volume of interest, and the treatment of the resulting 3D dataset are presented. The method is illustrated with data collected during the angiogenesis of blood vessels in a transgenic zebrafish embryo.
Asunto(s)
Embrión no Mamífero/ultraestructura , Neovascularización Fisiológica , Pez Cebra , Animales , Vasos Sanguíneos/ultraestructura , Tomografía con Microscopio Electrónico , Embrión no Mamífero/irrigación sanguínea , Resinas Epoxi/química , Imagenología Tridimensional , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Microscopía por Video , Microtomía , Adhesión en Plástico , Programas Informáticos , Fijación del TejidoRESUMEN
Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2(-/-)/Crb1(Rd8/RD8), Cx3cr1(-/-)/Crb1(Rd8/RD8) and CCl2(-/-)/Cx3cr1(-/-)/Crb1(Rd8/RD8) mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.
Asunto(s)
Quimiocina CCL2/inmunología , Receptores de Quimiocina/inmunología , Retina/patología , Degeneración Retiniana/inmunología , Degeneración Retiniana/patología , Animales , Receptor 1 de Quimiocinas CX3C , Quimiocina CCL2/genética , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteínas del Tejido Nervioso/genética , Receptores de Quimiocina/genética , Retina/inmunología , Retina/metabolismo , Degeneración Retiniana/genéticaRESUMEN
Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.
RESUMEN
The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.