RESUMEN
Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps, including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study because of their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here, we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion.
Asunto(s)
Fusión de Membrana , Proteínas SNARE , Membrana Celular , Difusión , Liposomas UnilamelaresRESUMEN
Interferometric scattering (iSCAT) microscopy is an emerging label-free technique optimized for the sensitive detection of nano-matter. Previous iSCAT studies have approximated the point spread function in iSCAT by a Gaussian intensity distribution. However, recent efforts to track the mobility of nanoparticles in challenging speckle environments and over extended axial ranges has necessitated a quantitative description of the interferometric point spread function (iPSF). We present a robust vectorial diffraction model for the iPSF in tandem with experimental measurements and rigorous FDTD simulations. We examine the iPSF under various imaging scenarios to understand how aberrations due to the experimental configuration encode information about the nanoparticle. We show that the lateral shape of the iPSF can be used to achieve nanometric three-dimensional localization over an extended axial range on the order of 10â µm either by means of a fit to an analytical model or calibration-free unsupervised machine learning. Our results have immediate implications for three-dimensional single particle tracking in complex scattering media.
RESUMEN
Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (1,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-1-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 µm is found to be free. In the case of functionalization with gold and 6-mercapto-1-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.
Asunto(s)
Diseño de Equipo/instrumentación , Membrana Dobles de Lípidos/química , Microscopía/instrumentación , Simulación por Computador , Difusión , Oro/química , Hexanoles/química , Nanopartículas del Metal/química , Método de Montecarlo , Tamaño de la Partícula , Fosfatidiletanolaminas/química , Porosidad , Compuestos de Silicona/química , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
Ion beams can be used to permanently bend and re-align nanowires after growth. We have irradiated ZnO nanowires with energetic ions, achieving bending and alignment in different directions. Not only the bending of single nanowires is studied in detail, but also the simultaneous alignment of large ensembles of ZnO nanowires. Computer simulations reveal how the bending is initiated by ion beam induced damage. Detailed structural characterization identifies dislocations to relax stresses and make the bending and alignment permanent, even surviving annealing procedures.
RESUMEN
Formation of a perfusable microvascular network (µVN) is critical for tissue engineering of solid organs. Stromal cells can support endothelial cell (EC) self-assembly into a µVN, but distinct stromal cell populations may play different roles in this process. Here we describe the differential effects that two widely used stromal cell populations, fibroblasts (FBs) and pericytes (PCs), have on µVN formation. We examined the effects of adding defined stromal cell populations on the self-assembly of ECs derived from human endothelial colony forming cells (ECFCs) into perfusable µVNs in fibrin gels cast within a microfluidic chamber. ECs alone failed to fully assemble a perfusable µVN. Human lung FBs stimulated the formation of EC-lined µVNs within microfluidic devices. RNA-seq analysis suggested that FBs produce high levels of hepatocyte growth factor (HGF). Addition of recombinant HGF improved while the c-MET inhibitor, Capmatinib (INCB28060), reduced µVN formation within devices. Human placental PCs could not substitute for FBs, but in the presence of FBs, PCs closely associated with ECs, formed a common basement membrane, extended microfilaments intercellularly, and reduced microvessel diameters. Different stromal cell types provide different functions in microvessel assembly by ECs. FBs support µVN formation by providing paracrine growth factors whereas PCs directly interact with ECs to modify microvascular morphology.
RESUMEN
Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of ß2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.
Asunto(s)
Aloinjertos/inmunología , Células Endoteliales/inmunología , Rechazo de Injerto/prevención & control , Proteínas Nucleares/genética , Ingeniería de Tejidos/métodos , Transactivadores/genética , Microglobulina beta-2/genética , Aloinjertos/irrigación sanguínea , Aloinjertos/provisión & distribución , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Progenitoras Endoteliales , Femenino , Sangre Fetal/citología , Técnicas de Inactivación de Genes , Rechazo de Injerto/sangre , Rechazo de Injerto/inmunología , Voluntarios Sanos , Humanos , Isoanticuerpos/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/genética , Ratones , Microvasos/citología , Microvasos/inmunología , Microvasos/trasplante , Proteínas Nucleares/inmunología , Trasplante de Órganos/efectos adversos , Trasplante de Órganos/métodos , Cultivo Primario de Células , Transactivadores/inmunología , Microglobulina beta-2/inmunologíaRESUMEN
The cell membrane forms a dynamic and complex barrier between the living cell and its environment. However, its in vivo studies are difficult because it consists of a high variety of lipids and proteins and is continuously reorganized by the cell. Therefore, membrane model systems with precisely controlled composition are used to investigate fundamental interactions of membrane components under well-defined conditions. Giant unilamellar vesicles (GUVs) offer a powerful model system for the cell membrane, but many previous studies have been performed in unphysiologically low ionic strength solutions which might lead to altered membrane properties, protein stability and lipid-protein interaction. In the present work, we give an overview of the existing methods for GUV production and present our efforts on forming single, free floating vesicles up to several tens of µm in diameter and at high yield in various buffer solutions with physiological ionic strength and pH.
RESUMEN
Although trapping and manipulation of small objects have been of interest for a range of applications and many clever techniques have been devised, new methods are still in great demand for handling different materials and geometries. Here, we report on an electrostatic trap that is created in an aqueous medium between the aperture of a nanopipette and a glass substrate without the need for external potentials. After a thorough characterization of the trapping conditions, we show that we can displace or release a particle at will. Furthermore, we demonstrate trapping and manipulation of nanoparticles and lipid vesicles attached to lipid bilayers, paving the way for controlled studies of forces and diffusion associated with biological membranes. We expect the technique to find interesting applications also in other areas such as optonanofluidics and plasmonics.
Asunto(s)
Vidrio/química , Membrana Dobles de Lípidos/química , Nanopartículas/química , Electricidad Estática , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Difusión , Interferometría/métodos , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica de Rastreo , Nanopartículas/ultraestructura , Tamaño de la Partícula , Termodinámica , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
Supported lipid bilayers have been studied intensively over the past two decades. In this work, we study the diffusion of single gold nanoparticles (GNPs) with diameter of 20 nm attached to GM1 ganglioside or DOPE lipids at different concentrations in supported DOPC bilayers. The indefinite photostability of GNPs combined with the high sensitivity of interferometric scattering microscopy (iSCAT) allows us to achieve 1.9 nm spatial precision at 1 ms temporal resolution, while maintaining long recording times. Our trajectories visualize strong transient confinements within domains as small as 20 nm, and the statistical analysis of the data reveals multiple mobilities and deviations from normal diffusion. We present a detailed analysis of our findings and provide interpretations regarding the effect of the supporting substrate and GM1 clustering. We also comment on the use of high-speed iSCAT for investigating diffusion of lipids, proteins, or viruses in lipid membranes with unprecedented spatial and temporal resolution.