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

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e16-07-0536 ◽

2017 ◽

Vol 28 (11) ◽

pp. 1507-1518 ◽

Cited By ~ 62

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.

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

Biochemical Journal ◽

10.1042/bj20091283 ◽

2009 ◽

Vol 424 (2) ◽

pp. 163-167 ◽

Cited By ~ 105

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.

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Observations on the Cell Biology of Tissue Factor in Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645213 ◽

1990 ◽

Vol 63 (02) ◽

pp. 298-302 ◽

Cited By ~ 18

Author(s):

Kiyoshi Andoh ◽

Kjell Sverre Pettersen ◽

Christiane Filion -Myklebust ◽

Hans Prydz

Keyword(s):

Endothelial Cells ◽

Protein Synthesis ◽

Plasma Membrane ◽

Tissue Factor ◽

Cell Biology ◽

Umbilical Vein ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells (HUVEC) are inducible for tissue factor (TF) activity in culture. Based on experiments using ECGF (4–20 µg/ml) with heparin (90 µg/ml), we obtained the followingresults: 1) In confluent HUVEC cultures, ECGF had essentially no influence on the levels of inducible TF. 2) In growing HUVEC cultures, ECGF reduced the TF response shortly after seeding but full response was regained when cells were kept confluent for 2–3 days. 3) Although secondary cultures responded best to TF induction in the absence of ECGF, the response was essentially equal over at least 8 passages in the prcgcncc of ECGF 1) Of total cellular TF induced in HUVEC, about 25% was available on the surface, and less than 4% was released with theshed plasma membrane vesicles. The proportion of total TF a>ctivity available on the surface of mtact cells wasnot influenced by the presence of ECGF 5) T½, for the decay of TF activity induced was 8.3—9.5 h, whereas in HUVEC when protein synthesis was blocked afterIF induction a T½ of about 30 h was found.

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

10.1101/2020.12.22.422352 ◽

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

10.1101/076109 ◽

2016 ◽

Cited By ~ 3

Author(s):

Falk Schneider ◽

Mathias P Clausen ◽

Dominic Waithe ◽

Thomas Koller ◽

Gunes Ozhan ◽

...

Keyword(s):

Plasma Membrane ◽

High Mobility ◽

Super Resolution ◽

Membrane Vesicles ◽

Correlation Spectroscopy ◽

Diffusion Regime ◽

Plasma Membrane Vesicles ◽

Intact Cells ◽

Actin Cortex ◽

Hindered Diffusion

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling. These have been suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access the sub-diffraction diffusion regime of different fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the cellular plasma membrane, and compare it to the diffusion regime of these molecules in cell-derived actin-free giant plasma membrane vesicles (GPMVs). We show that phospholipids and sphingomyelin, which undergo hindered diffusion in the live cell membrane, diffuse freely in the GPMVs. In contrast to sphingomyelin, which is transiently trapped on molecular-scale complexes in intact cells, diffusion of the ganglioside lipid GM1 suggests transient incorporation into nanodomains, which is less influenced by the actin cortex. Finally, our data on GPI-APs indicate two molecular pools in living cells, one pool showing high mobility with trapped and compartmentalized diffusion, and the other forming immobile clusters both of which disappear in GPMVs. Our data underlines the crucial role of the actin cortex in maintaining hindered diffusion modes of most but not all membrane molecules.

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