Search Results
Fluorescence Microscopy of the HIV-1 Envelope
Human immunodeficiency virus (HIV) infection constitutes a major health and social issue worldwide. HIV infects cells by fusing its envelope with the target cell plasma membrane. This process is mediated by the viral Env glycoprotein and depends on the envelope lipid composition. Fluorescent microscopy has been employed to investigate the envelope properties, and the processes of viral assembly and fusion, but the application of this technique to the study of HIV is still limited by a number of factors, such as the small size of HIV virions or the difficulty to label the envelope components. Here, we review fluorescence imaging studies of the envelope lipids and proteins, focusing on labelling strategies and model systems.
Comparison of Multiscale Imaging Methods for Brain Research
A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections.
Immune mobilising T cell receptors redirect polyclonal CD8+ T cells in chronic HIV infection to form immunological synapses
T cell exhaustion develops in human immunodeficiency virus (HIV) infection due to chronic viral antigenic stimulation. This adaptive response primarily affects virus-specific CD8+ T cells, which may remain dysfunctional despite viral load-reducing antiretroviral therapy; however, abnormalities may also be evident in non-HIV-specific populations. Both could limit the efficacy of cell therapies against viral reservoirs. Here, we show that bulk (polyclonal) CD8+ T cells from people living with HIV (PLWH) express proposed markers of dysfunctional HIV-specific T cells at high levels yet form lytic immunological synapses (IS) and eliminate primary resting infected (HIV Gaglo) CD4+ T cells, when redirected by potent bispecific T cell-retargeting molecules, Immune mobilising monoclonal T cell receptors (TCR) Against Virus (ImmTAV). While PLWH CD8+ T cells are functionally impaired when compared to CD8+ T cells from HIV-naïve donors, ImmTAV redirection enables them to eliminate Gaglo CD4+ T cells that are insensitive to autologous HIV-specific cytolytic T cells. ImmTAV molecules may therefore be able to target HIV reservoirs, which represent a major barrier to a cure.
Super-resolution RESOLFT microscopy of lipid bilayers using a fluorophore-switch dyad
Dyads consisting of a photochromic switch covalently linked to a fluorescent dye allow the emission from the dye to be controlled by reversible photoisomerization of the switch; one form of the switch quenches fluorescence by accepting energy from the dye. Here we investigate the use of dyads of this type for super-resolution imaging of lipid bilayers. Giant unilamellar vesicles stained with the dyads were imaged with about a two-fold resolution-enhancement compared with conventional confocal microscopy. This was achieved by exciting the fluorophore at 594 nm, using a switch activated by violet and red light (405/640 nm). This journal is © The Royal Society of Chemistry.
Object detection networks and augmented reality for cellular detection in fluorescence microscopy
Object detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines. © 2020 Waithe et al.
Flotillin-Dependent Membrane Microdomains Are Required for Functional Phagolysosomes against Fungal Infections
Schmidt el al. show that lipid rafts in phagolysosomal membranes of macrophages depend on flotillins. Lipid rafts are required for assembly of vATPase and NADPH oxidase. Conidia of the human-pathogenic fungus Aspergillus fumigatus dysregulate assembly of flotillin-dependent lipid rafts in the phagolysosomal membrane and can thereby escape phagolysosomal digestion. © 2020 The Author(s)Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions and that inhibition of Ca2+-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity. © 2020 The Author(s)
Super-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells
The ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions.
Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments
The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes. © 2020 The Author(s)Rujas et al. describe the site-selective chemical modification of antibodies to improve the molecular recognition of epitopes at membrane surfaces. The modification using aromatic compounds dramatically enhanced the virus neutralization potency and native antigen binding efficiency of HIV-1 antibodies directed against the membrane-embedded MPER epitope. © 2020 The Author(s)
Intracellular Photophysics of an Osmium Complex bearing an Oligothiophene Extended Ligand
This contribution describes the excited-state properties of an Osmium-complex when taken up into human cells. The complex 1 [Os(bpy)2(IP-4T)](PF6)2 with bpy=2,2′-bipyridine and IP-4T=2-{5′-[3′,4′-diethyl-(2,2′-bithien-5-yl)]-3,4-diethyl-2,2′-bithiophene}imidazo[4,5-f][1,10]phenanthroline) can be discussed as a candidate for photodynamic therapy in the biological red/NIR window. The complex is taken up by MCF7 cells and localizes rather homogeneously within in the cytoplasm. To detail the sub-ns photophysics of 1, comparative transient absorption measurements were carried out in different solvents to derive a model of the photoinduced processes. Key to rationalize the excited-state relaxation is a long-lived 3ILCT state associated with the oligothiophene chain. This model was then tested with the complex internalized into MCF7 cells, since the intracellular environment has long been suspected to take big influence on the excited state properties. In our study of 1 in cells, we were able to show that, though the overall model remained the same, the excited-state dynamics are affected strongly by the intracellular environment. Our study represents the first in depth correlation towards ex-vivo and in vivo ultrafast spectroscopy for a possible photodrug. © 2020 The Authors. Published by Wiley-VCH GmbH
The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1
Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan- binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration. © 2020 Stanly et al.