4 results
Search Results
Now showing 1 - 4 of 4
- ItemFlotillin-Dependent Membrane Microdomains Are Required for Functional Phagolysosomes against Fungal Infections(Maryland Heights, MO : Cell Press, 2020) Schmidt, Franziska; Thywißen, Andreas; Goldmann, Marie; Cunha, Cristina; Cseresnyés, Zoltán; Schmidt, Hella; Rafiq, Muhammad; Galiani, Silvia; Gräler, Markus H.; Chamilos, Georgios; Lacerda, João; Campos, António, Jr.; Eggeling, Christian; Figge, Marc Thilo; Heinekamp, Thorsten; Filler, Scott G.; Carvalho, Agostinho; Brakhage, Axel A.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)
- ItemSuper-resolution RESOLFT microscopy of lipid bilayers using a fluorophore-switch dyad(Cambridge : RSC, 2020) Frawley, Andrew T.; Wycisk, Virginia; Xiong, Yaoyao; Galiani, Silvia; Sezgin, Erdinc; Urbančič, Iztok; Vargas Jentzsch, Andreas; Leslie, Kathryn G.; Eggeling, Christian; Anderson, Harry L.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.
- ItemBackground Reduction in STED-FCS Using a Bivortex Phase Mask(Washington, DC : ACS Publications, 2020) Barbotin, Aurélien; Urbančič, Iztok; Galiani, Silvia; Eggeling, Christian; Booth, MartinFluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics in living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes on the nanoscale in living cells. In two-dimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volume, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern created by a bivortex phase mask reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS-based investigations of 3D diffusion on the subdiffraction scale. Copyright © 2020 American Chemical Society.
- Itemz-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics(Bethesda, Md. : Biophysical Soc., 2020) Barbotin, Aurélien; Urbančič, Iztok; Galiani, Silvia; Eggeling, Christian; Booth, Martin; Sezgin, ErdincSuper-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively. © 2020 Biophysical Society