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- ItemNanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer(Washington, DC : ACS Publ., 2018-7-24) Urbančič, Iztok; Garvas, Maja; Kokot, Boštjan; Majaron, Hana; Umek, Polona; Cassidy, Hilary; Škarabot, Miha; Schneider, Falk; Galiani, Silvia; Arsov, Zoran; Koklic, Tilen; Matallanas, David; Čeh, Miran; Muševič, Igor; Eggeling, Christian; Štrancar, JanezAlthough the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane’s disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.
- 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.
- ItemMechanical properties of plasma membrane vesicles correlate with lipid order, viscosity and cell density(Berlin : Nature Publishing, 2019) Steinkühler, Jan; Sezgin, Erdinc; Urbančič, Iztok; Eggeling, Christian; Dimova, RumianaRegulation of plasma membrane curvature and composition governs essential cellular processes. The material property of bending rigidity describes the energetic cost of membrane deformations and depends on the plasma membrane molecular composition. Because of compositional fluctuations and active processes, it is challenging to measure it in intact cells. Here, we study the plasma membrane using giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. We show that the bending rigidity of plasma membranes under varied conditions is correlated to readout from environment-sensitive dyes, which are indicative of membrane order and microviscosity. This correlation holds across different cell lines, upon cholesterol depletion or enrichment of the plasma membrane, and variations in cell density. Thus, polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Additionally, our results allow for identifying synthetic membranes with a few well defined lipids as optimal plasma membrane mimetics.
- ItemHigh photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy(Bristol : IOP Publ., 2020) Schneider, Falk; Hernandez-Varas, Pablo; Lagerholm, B. Christoffer; Shrestha, Dilip; Sezgin, Erdinc; Roberti, M. Julia; Ossato, Giulia; Hecht, Frank; Eggeling, Christian; Urbančič, IztokProbing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 µm. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells. © The Author(s). Published by IOP Publishing Ltd.
- 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
- ItemAggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells(Chichester : Wiley, 2021) Urbančič, Iztok; Schiffelers, Lisa; Jenkins, Edward; Gong, Weijian; Santos, Ana Mafalda; Schneider, Falk; O'Brien-Ball, Caitlin; Vuong, Mai Tuyet; Ashman, Nicole; Sezgin, Erdinc; Eggeling, ChristianTo disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.
- ItemAdaptive optics allows STED-FCS measurements in the cytoplasm of living cells(Washington D.C. : Optical Society of America, 2019) Barbotin, Aurélien; Galiani, Silvia; Urbančič, Iztok; Eggeling, Christian; Booth, Martin J.Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (z) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells.
- ItemLipid Composition but Not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles(Basel : MDPI, 2018) Urbančič, Iztok; Brun, Juliane; Shrestha, Dilip; Waithe, Dominic; Eggeling, Christian; Chojnacki, JakubHuman Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from which it buds out. Previous studies have shown that the HIV-1 envelope is an environment of very low mobility, with the diffusion of incorporated proteins two orders of magnitude slower than in the plasma membrane. One of the reasons for this difference is thought to be the HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing, which has, until now, been difficult to assess experimentally. To further refine the model of the molecular mobility on the HIV-1 surface, we herein investigated the relative importance of membrane composition and curvature in simplified model membrane systems, large unilamellar vesicles (LUVs) of different lipid compositions and sizes (0.1–1 µm), using super-resolution stimulated emission depletion (STED) microscopy-based fluorescence correlation spectroscopy (STED-FCS). Establishing an approach that is also applicable to measurements of molecule dynamics in virus-sized particles, we found, at least for the 0.1–1 µm sized vesicles, that the lipid composition and thus membrane rigidity, but not the curvature, play an important role in the decreased molecular mobility on the vesicles’ surface. This observation suggests that the composition of the envelope rather than the particle geometry contributes to the previously described low mobility of proteins on the HIV-1 surface. Our vesicle-based study thus provides further insight into the dynamic properties of the surface of individual HIV-1 particles, as well as paves the methodological way towards better characterisation of the properties and function of viral lipid envelopes in general.