Creating Supported Plasma Membrane Bilayers Using Acoustic Pressure

Model membrane systems are essential tools for the study of biological processes in a simplified setting to reveal the underlying physicochemical principles. As cell-derived membrane systems, giant plasma membrane vesicles (GPMVs) constitute an intermediate model between live cells and fully artificial structures. Certain applications, however, require planar membrane surfaces. Here, we report a new approach for creating supported plasma membrane bilayers (SPMBs) by bursting cell-derived GPMVs using ultrasound within a microfluidic device. We show that the mobility of outer leaflet molecules is preserved in SPMBs, suggesting that they are accessible on the surface of the bilayers. Such model membrane systems are potentially useful in many applications requiring detailed characterization of plasma membrane dynamics.

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Creating supported plasma membrane bilayers using acoustic pressure

10.1101/2020.01.20.912840 ◽

2020 ◽

Author(s):

Erdinc Sezgin ◽

Dario Carugo ◽

Ilya Levental ◽

Eleanor Stride ◽

Christian Eggeling

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Membrane Dynamics ◽

Model Membrane ◽

Membrane Systems ◽

Plasma Membrane Vesicles ◽

Outer Leaflet ◽

Membrane Surfaces ◽

Membrane Bilayers ◽

Novel Approach

AbstractModel membrane systems are essential tools for biology, enabling study of biological processes in a simplified setting to reveal the underlying physicochemical principles. As cell-derived membrane systems, giant plasma membrane vesicles (GPMVs) constitute an intermediate model between native cellular plasma and artificial membranes. Certain applications, however, require planar membrane surfaces. Here, we report a novel approach for creating supported plasma membrane bilayers (SPMBs) by bursting cell-derived GPMVs using an ultrasonic pressure field generated within an acoustofluidic device. We show that the mobility of outer leaflet molecules is preserved in SPMBs, suggesting that they are accessible on the surface of the bilayers. Such model membrane systems will be useful for many applications requiring detailed characterization of plasma membrane dynamics.

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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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