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 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 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.

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.

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 Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kaushik Inamdar ◽  
Feng-Ching Tsai ◽  
Rayane Dibsy ◽  
Aurore de Poret ◽  
John Manzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Particle Assembly ◽  
Gag Protein ◽  
Cell Plasma ◽  
Curved Membranes ◽  
Hiv 1

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. SiRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

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 Resolving the Effects of Nanoscale Membrane Curvature on Lipid Mobility

2017 ◽  
Author(s):  
Abir Maarouf Kabbani ◽  
Xinxin Woodward ◽  
Christopher V. Kelly
Keyword(s):  
Lipid Bilayers ◽  
Super Resolution ◽  
Membrane Curvature ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Optical Diffraction ◽  
Membrane Behavior ◽  
Lipid Mobility ◽  
Experimental Resolution ◽  
Membrane Budding

The biophysical consequences of nanoscale curvature have been challenging to resolve due to size-dependent membrane behavior and the experimental resolution limits imposed by optical diffraction. Recent advances in nanoengineering and super-resolution techniques have enabled new capabilities for creating and observing curvature. In particular, draping supported lipid bilayers over lithographically patterned substrates provides a model system for endocytic pits. The experiments and simulations presented below describe the possible detection of membrane curvature through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT), and polarized localization microscopy (PLM). FRAP and FCS depend on diffraction-limited illumination and detection. In particular, a simulation of FRAP shows no effects on lipids diffusion due to a 50 nm diameter membrane bud at any stage in the budding process. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was amplified with curvature-dependent lipid mobility changes. However, PLM and SPT achieve sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of curvature on lipid behavior have been resolved.

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.

Drug Permeation through Biological Barriers

Drug Delivery ◽  
2001 ◽  
Author(s):  
W. Mark Saltzman
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Lipid Membranes ◽  
Lipid Membrane ◽  
Average Molecular Weight ◽  
Transport Proteins ◽  
Cross Sectional ◽  
Tissue Properties ◽  
Experimental Systems ◽  
Outer Leaflet

In multicellular organisms, thin lipid membranes serve as semipermeable barriers between aqueous compartments. The plasma membrane of the cell separates the cytoplasm from the extracellular space; endothelial cell membranes separate the blood within the vascular space from the rest of the tissue. Properties of the lipid membrane are critically important in regulating the movement of molecules between these aqueous spaces. While certain barrier properties of membranes can be attributed to the lipid components, accessory molecules within the cell membrane—particularly transport proteins and ion channels—control the rate of permeation of many solutes. Transport proteins permit the cell to regulate the composition of its intracellular environment in response to extracellular conditions. The relationship between membrane structure, membrane function, and cell physiology is an area of active, ongoing study. Our interest here is practical: what are the basic mechanisms of drug movement through membranes and how can one best predict the rate of permeation of an agent through a membrane barrier? To answer that question, this section presents rates of permeation measured in some common experimental systems and models of membrane permeation that can be used for prediction. The external surface of the plasma membrane carries a carbohydrate-rich coat called the glycocalyx; charged groups in the glycocalyx, which are provided principally by carbohydrates containing sialic acid, cause the surface to be negatively charged. On average, the plasma membrane of human cells contains, by mass, 50% protein, 45% lipid, and 5% carbohydrate. Given the mass ratio of protein to lipid is ~ 1 : 1, and assuming reasonable values for the average molecular weight and cross-sectional area for each type of molecule (50 × Mw,lipid = Mw,protein; Alipid = 50 Å2 and Aprotein = 1,000 Å2), the area fraction of protein on a typical membrane is ~ 33%. The lipid composition varies in membranes from different cells depending on the type of cell and its function. In addition, the outermost monolayer of lipids, called the outer leaflet, has a different lipid composition from the inner leaflet.

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