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- 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.
- ItemMolecular recognition of the native HIV-1 MPER revealed by STED microscopy of single virions(Berlin : Nature Publishing, 2019) Carravilla, Pablo; Chojnacki, Jakub; Rujas, Edurne; Insausti, Sara; Largo, Eneko; Waithe, Dominic; Apellaniz, Beatriz; Sicard, Taylor; Julien, Jean-Philippe; Eggeling, Christian; Nieva, José L.Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.
- ItemReconstitution of immune cell interactions in free-standing membranes(Cambridge : Company of Biologists, 2019) Jenkins, Edward; Santos, Ana M.; Felce, James H; Hatherley, Deborah; Dustin, Michael L.; Davis, Simon J.; Eggeling, Christian; Sezgin, ErdincThe spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be ‘dialled-in’ as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable.The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be ‘dialled-in’ as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable.
- ItemThe 2015 super-resolution microscopy roadmap(Bristol : IOP Publ., 2015) Hell, Stefan W.; Sahl, Steffen J.; Bates, Mark; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J.; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Jakobs, Stefan; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J.; Eggeling, Christian; Klenerman, David; Willig, Katrin I.; Vicidomini, Giuseppe; Castello, Marco; Diaspro, Alberto; Cordes, ThorbenFar-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of 'super-resolution' far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact.
- ItemMore Favorable Palmitic Acid Over Palmitoleic Acid Modification of Wnt3 Ensures Its Localization and Activity in Plasma Membrane Domains(Lausanne : Frontiers Media, 2019) Azbazdar, Yagmur; Ozalp, Ozgun; Sezgin, Erdinc; Veerapathiran, Sapthaswaran; Duncan, Anna L.; Sansom, Mark S.P.; Eggeling, Christian; Wohland, Thorsten; Karaca, Ezgi; Ozhan, GunesWhile the lateral organization of plasma membrane components has been shown to control binding of Wnt ligands to their receptors preferentially in the ordered membrane domains, the role of posttranslational lipid modification of Wnt on this selective binding is unknown. Here, we identify that the canonical Wnt is presumably acylated by palmitic acid, a saturated 16-carbon fatty acid, at a conserved serine residue. Acylation of Wnt3 is dispensable for its secretion and binding to Fz8 while it is essential for Wnt3's proper binding and domain-like diffusion in the ordered membrane domains. We further unravel that non-palmitoylated Wnt3 is unable to activate Wnt/β-catenin signaling either in zebrafish embryos or in mammalian cells. Based on these results, we propose that the lipidation of canonical Wnt, presumably by a saturated fatty acid, determines its competence in interacting with the receptors in the appropriate domains of the plasma membrane, ultimately keeping the signaling activity under control. © Copyright © 2019 Azbazdar, Ozalp, Sezgin, Veerapathiran, Duncan, Sansom, Eggeling, Wohland, Karaca and Ozhan.
- 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.
- ItemHIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly(Washington D.C. : AAAS, 2019) Favard, C.; Chojnacki, Jakub; Merida, P.; Yandrapalli, N.; Mak, J.; Eggeling, Christian; Muriaux, D.HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly.
- ItemNanoscale dynamics of cholesterol in the cell membrane(Bethesda, Md. : ASBMB Publications, 2019) Pinkwart, Kerstin; Schneider, Falk; Lukoseviciute, Martyna; Sauka-Spengler, Tatjana; Lyman, Edward; Eggeling, Christian; Sezgin, ErdincCholesterol constitutes ~30-40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane. © 2019 Pinkwart et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.
- ItemSuper-resolution fluorescence microscopy studies of human immunodeficiency virus(London : BioMed Central, 2018) Chojnacki, Jeremy; Eggeling, ChristianSuper-resolution fluorescence microscopy combines the ability to observe biological processes beyond the diffraction limit of conventional light microscopy with all advantages of the fluorescence readout such as labelling specificity and non-invasive live-cell imaging. Due to their subdiffraction size (< 200 nm) viruses are ideal candidates for super-resolution microscopy studies, and Human Immunodeficiency Virus type 1 (HIV-1) is to date the most studied virus by this technique. This review outlines principles of different super-resolution techniques as well as their advantages and disadvantages for virological studies, especially in the context of live-cell imaging applications. We highlight the findings of super-resolution based HIV-1 studies performed so far, their contributions to the understanding of HIV-1 replication cycle and how the current advances in super-resolution microscopy may open new avenues for future virology research.
- 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.