scholarly journals Lipid Composition but not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 415 ◽  
Author(s):  
Iztok Urbančič ◽  
Juliane Brun ◽  
Dilip Shrestha ◽  
Dominic Waithe ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Molecular Mobility ◽  
Lipid Membrane ◽  
Dynamic Properties ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Membrane Composition ◽  
Hiv 1

Human 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.

scholarly journals Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

2018 ◽  
Author(s):  
Iztok Urbančič ◽  
Juliane Brun ◽  
Dilip Shrestha ◽  
Dominic Waithe ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Molecular Mobility ◽  
Lipid Membrane ◽  
Dynamic Properties ◽  
Super Resolution ◽  
Membrane Curvature ◽  
Correlation Spectroscopy ◽  
Membrane Composition ◽  
Hiv 1

AbstractHuman Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2–1 μm) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

scholarly journals Super-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 608
Author(s):  
Jakub Chojnacki ◽  
Christian Eggeling
Keyword(s):  
Molecular Dynamics ◽  
Plasma Membrane ◽  
Molecular Mobility ◽  
Virus Assembly ◽  
Methodological Approach ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Model Systems ◽  
Hiv 1

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.

Super-resolution optical microscopy of lipid plasma membrane dynamics

2015 ◽  
Vol 57 ◽  
pp. 69-80 ◽  
Author(s):  
Christian Eggeling
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Molecular Diffusion ◽  
Super Resolution ◽  
Optical Microscope ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Diffusion Dynamics ◽  
Lipid Protein Interactions

Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.

scholarly journals HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

2019 ◽  
Vol 5 (10) ◽  
pp. eaaw8651 ◽  
Author(s):  
C. Favard ◽  
J. Chojnacki ◽  
P. Merida ◽  
N. Yandrapalli ◽  
J. Mak ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Virus Assembly ◽  
Super Resolution ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Gag Protein ◽  
Lipid Dynamics ◽  
Infected Cells ◽  
Lipid Environment ◽  
Hiv 1

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.

scholarly journals HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

2019 ◽  
Author(s):  
C. Favard ◽  
J. Chojnacki ◽  
P. Merida ◽  
N. Yandrapalli ◽  
J. Mak ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Virus Assembly ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Gag Protein ◽  
Lipid Dynamics ◽  
Cell Plasma ◽  
Infected Cells ◽  
Lipid Environment ◽  
Hiv 1

HIV-1 Gag protein self-assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly the phosphatidylinositol (4, 5) bisphosphate, 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 was determined using super-resolution STED microscopy coupled with scanning Fluorescence Correlation Spectroscopy in living T 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 show that Gag is the main driving force to restrict PI(4,5)P2 and cholesterol mobility at the cell plasma membrane. This is first direct evidence showing that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol, as a membrane nano-platform for virus assembly.

scholarly journals The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

2020 ◽  
Vol 295 (15) ◽  
pp. 5036-5050 ◽  
Author(s):  
Tess A. Stanly ◽  
Marco Fritzsche ◽  
Suneale Banerji ◽  
Dilip Shrestha ◽  
Falk Schneider ◽  
...  
Keyword(s):  
Lymphatic Vessel ◽  
Leukocyte Adhesion ◽  
Lymphatic Vessels ◽  
Super Resolution ◽  
Direct Interaction ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Actin Network ◽  
Cortical Actin ◽  
Limited Mobility

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.

Effects of membrane lipid and fluidity modifications on HIV-1 infectibility of primate lymphocytes in vitro

1990 ◽  
Vol 10 (3) ◽  
pp. 263-270 ◽  
Author(s):  
J. Pascal Zimmer ◽  
Hans A. Lehr ◽  
Christoph Hübner ◽  
Stephan G. Lindner ◽  
Ralf Ramsperger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fluidity ◽  
Lipid Composition ◽  
Membrane Lipid ◽  
Serum Factors ◽  
Membrane Lipid Composition ◽  
Plasma Membrane Lipid ◽  
Hiv 1

Although most non-human primates, except the chimpanzee and the gibbon in vivo are not infectible by HIV-1, lymphocytes of several of these species can be infected by HIV-1 in vitro.In order to investigate whether the in vitro infectibility of primate lymphocytes might be attributed to plasma membrane adaptation processes or to serum factors, we compared HIV-1 infectibility of cultivated peripheral blood lymphocytes of macaques and of baboons on day one and on day ten of cultivation. These data were correlated to plasma membrane lipid composition and membrane fluidity.We found a correlation between increased HIV-1 in vitro infectibility and changes in plasma membrane lipid composition resulting in decreased membrane fluidity of cultured primate lymphocytes.

scholarly journals STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

2021 ◽  
Author(s):  
Pablo Carravilla ◽  
Anindita Dasgupta ◽  
Gaukhar Zhurgenbayeva ◽  
Dmytro I. Danylchuk ◽  
Andrey S. Klymchenko ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Spatial Resolution ◽  
Super Resolution ◽  
Membrane Dynamics ◽  
Live Cell ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Temporal And Spatial ◽  
Polarity Sensitive

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high temporal and spatial resolution and long acquisition times, that also allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolution, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dyes is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

scholarly journals Polarity sensitive probes for super resolution STED microscopy

2017 ◽  
Author(s):  
E Sezgin ◽  
F Schneider ◽  
V Zilles ◽  
E Garcia ◽  
D Waithe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Membrane ◽  
Membrane Lipids ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Molecular Organization ◽  
Live Cells ◽  
Sted Microscopy ◽  
Two Photon Microscopy ◽  
Polarity Sensitive

AbstractThe lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.

scholarly journals Adaptive optics allows 3D STED-FCS measurements in the cytoplasm of living cells

2019 ◽  
Author(s):  
Aurélien Barbotin ◽  
Silvia Galiani ◽  
Iztok Urbančič ◽  
Christian Eggeling ◽  
Martin Booth
Keyword(s):  
Adaptive Optics ◽  
Molecular Diffusion ◽  
Three Dimensional ◽  
Super Resolution ◽  
Correction Method ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Optical Aberrations ◽  
Extreme Sensitivity

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 (3D) 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.

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