scholarly journals Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles

2009 ◽  
Vol 424 (2) ◽  
pp. 163-167 ◽  
Author(s):  
Ilya Levental ◽  
Fitzroy J. Byfield ◽  
Pramit Chowdhury ◽  
Feng Gai ◽  
Tobias Baumgart ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Membrane Vesicles ◽  
Correlation Spectroscopy ◽  
Model Systems ◽  
Live Cells ◽  
Lipid Phase ◽  
Plasma Membrane Vesicles ◽  
Liquid Ordered ◽  
Lipid Phase Separation

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

scholarly journals Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy

2022 ◽  
Author(s):  
Nirmalya Bag ◽  
Erwin London ◽  
David A Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Fluorescence Correlation Spectroscopy ◽  
Immunoglobulin E ◽  
Correlation Spectroscopy ◽  
Model Systems ◽  
Live Cells ◽  
Membrane Diffusion ◽  
Fluorescence Correlation ◽  
The Impact

Plasma membrane hosts numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation of the plasma membrane is emerging as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the co-existence liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It is recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy we choose two well-established model systems for transbilayer interactions: crosslinking by multivalent antigen of immunoglobulin E bound to receptor FcεRI, and crosslinking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.

scholarly journals Biomembrane liquid–liquid phase separation and detergent resistance: a relationship strengthened

2009 ◽  
Vol 424 (2) ◽  
pp. e5-e6 ◽  
Author(s):  
David Holowka
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Liquid Phase ◽  
Lipid Rafts ◽  
Membrane Vesicles ◽  
Liquid Phase Separation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Acyl Chains ◽  
Liquid Liquid Phase Separation

Since evidence first appeared for ‘detergent-resistant membranes’ in the early to mid-1990s, cell biologists from a wide spectrum of biological sciences have been intrigued by the functional relevance of this indication of membrane heterogeneity, commonly referred to as ‘lipid rafts’. Model membrane studies revealed that these lipid rafts are related to the more ordered liquid phase that forms in a ternary mixture of cholesterol with a phospholipid containing saturated acyl chains and one with unsaturated acyl chains. Giant plasma membrane vesicles that pinch off from cells undergo similar liquid–liquid phase separation as ternary model membranes, and have provided an experimental bridge between these and intact cells. The study by Levental et al. in this issue of the Biochemical Journal provides new insights into the relationship between liquid–liquid phase separation in these plasma membrane vesicles and detergent-resistance of cellular lipid rafts.

scholarly journals Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

2007 ◽  
Vol 104 (9) ◽  
pp. 3165-3170 ◽  
Author(s):  
T. Baumgart ◽  
A. T. Hammond ◽  
P. Sengupta ◽  
S. T. Hess ◽  
D. A. Holowka ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Large Scale ◽  
Membrane Vesicles ◽  
Fluid Phase ◽  
Plasma Membrane Vesicles ◽  
Separation Of Proteins

scholarly journals Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes

2020 ◽  
Author(s):  
Daniel Beckers ◽  
Dunja Urbancic ◽  
Erdinc Sezgin
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Membrane Vesicles ◽  
Theoretical Models ◽  
Correlation Spectroscopy ◽  
Suitable Model ◽  
Biophysical Properties ◽  
Plasma Membrane Vesicles ◽  
Free Standing ◽  
Lipid Packing

AbstractMembrane models have allowed for precise study of the plasma membrane’s biophysical properties, helping to unravel both structural and dynamic motifs within cell biology. Free standing and supported bilayer systems are popular models to reconstitute the membrane related processes. Although it is well-known that each have their advantages and limitations, comprehensive comparison of their biophysical properties is still lacking. Here, we compare the diffusion and lipid packing in giant unilamellar vesicles, planar and spherical supported membranes and cell-derived giant plasma membrane vesicles. We apply florescence correlation spectroscopy, spectral imaging and super-resolution STED-FCS to study the diffusivity, lipid packing and nanoscale architecture of these membrane systems, respectively. Our data show that lipid packing and diffusivity is tightly correlated in free-standing bilayers. However, nanoscale interactions in the supported bilayers cause deviation from this correlation. This data is essential to develop accurate theoretical models of the plasma membrane and will serve as a guideline for suitable model selection in future studies to reconstitute biological processes.

scholarly journals Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

2017 ◽  
Vol 28 (11) ◽  
pp. 1507-1518 ◽  
Author(s):  
Falk Schneider ◽  
Dominic Waithe ◽  
Mathias P. Clausen ◽  
Silvia Galiani ◽  
Thomas Koller ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
High Mobility ◽  
Membrane Vesicles ◽  
Free Cell ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Live Cell ◽  
Plasma Membrane Vesicles ◽  
Hindered Diffusion

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signaling and are suggested to be strongly associated with the actin cytoskeleton. Here we use superresolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live-cell plasma membrane and in actin cytoskeleton–free, cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids is abolished in the GPMVs, whereas transient nanodomain incorporation of ganglioside lipid GM1 is apparent in both the live-cell membrane and GPMVs. For GPI-APs, we detect two molecular pools in living cells; one pool shows high mobility with transient incorporation into nanodomains, and the other pool forms immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules and highlight a powerful experimental approach to decipher specific influences on molecular plasma membrane dynamics.

scholarly journals Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains

2021 ◽  
Vol 118 (9) ◽  
pp. e2025343118
Author(s):  
Jongyun Myeong ◽  
Cheon-Gyu Park ◽  
Byung-Chang Suh ◽  
Bertil Hille
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Membrane Vesicles ◽  
Membrane Lipid ◽  
Phosphoinositide Metabolism ◽  
Protein Markers ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Liquid Ordered

Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

scholarly journals Phase Partitioning of Peptide Anchors in Plasma Membrane Vesicles Predicts Their Recruitment to B Cell Receptor Clusters in Live Cells

2019 ◽  
Vol 116 (3) ◽  
pp. 164a-165a
Author(s):  
Sarah A. Shelby ◽  
Ivan C. Serrano ◽  
Kandice R. Levental ◽  
Ilya Levental ◽  
Sarah L. Veatch
Keyword(s):  
Plasma Membrane ◽  
B Cell ◽  
Membrane Vesicles ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Live Cells ◽  
Plasma Membrane Vesicles ◽  
Phase Partitioning ◽  
Receptor Clusters

scholarly journals Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane

2001 ◽  
Vol 1512 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Saame Raza Shaikh ◽  
Alfred C Dumaual ◽  
Laura J Jenski ◽  
William Stillwell
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Phospholipid Bilayers ◽  
Lipid Phase ◽  
Lipid Phase Separation

scholarly journals Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells

2020 ◽  
Author(s):  
Iztok Urbančič ◽  
Lisa Schiffelers ◽  
Edward Jenkins ◽  
Weijian Gong ◽  
Ana Mafalda Santos ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Cell Receptor ◽  
Membrane Lipid ◽  
Correlation Spectroscopy ◽  
Membrane Properties ◽  
Plasma Membrane Vesicles ◽  
Aggregation State ◽  
Lipid Order

The quest for understanding of numerous vital membrane-associated cellular processes, such as signalling, has largely focussed on the spatiotemporal orchestration and reorganisation of the identified key proteins, including their binding and aggregation. Despite strong indications of the involvement of membrane lipid heterogeneities, historically often termed lipid rafts, their roles in many processes remain controversial and mechanisms elusive. Taking activation of T lymphocytes as an example, we here investigate membrane properties around the key proteins − in particular the T cell receptor (TCR), its main kinase Lck, and phosphatase CD45. We determine their partitioning and co-localisation in passive cell-derived model membranes (i.e. giant plasma-membrane vesicles, GPMVs), and explore their mobility and local lipid order in live Jurkat T cells using fluorescence correlation spectroscopy and spectral imaging with polarity-sensitive membrane probes. We find that upon aggregation and partial immobilisation, the TCR changes its preference towards more ordered lipid environments, which can in turn passively recruit Lck. We observe similar aggregation-induced local membrane ordering and recruitment of Lck also by CD45, as well as by a membrane protein of antigen-presenting cells, CD86, which is not supposed to interact with Lck directly. This highlights the involvement of lipid-mediated interactions and suggests that the cellular membrane is poised to modulate the frequency of protein encounters according to their aggregation state and alterations of their mobility, e.g. upon ligand binding.

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