Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins

Abstract

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Constructing Supported Cell Membranes with Controllable Orientation

“…We have previously shown that we can rupture GPMVs onto a glass surface to form supported plasma membranes, and the immunostaining results suggested that the supported plasma membranes have their intracellular sides facing the bulk solution 19 . In order to study either side of a supported plasma membrane, we intended to develop a blotting technique to reverse the membrane orientation.…”

“…Previous studies have shown that it is challenging to control the insertion orientation of membrane proteins while reconstituting the proteins into artificial lipid membranes 6,7,17,18 . Therefore, many studies have obtained cell membrane vesicles directly from cells as these membrane vesicles should preserve the native orientation of membrane proteins 12–16,19 .…”

“…Several studies have further deposited such cell membrane vesicles on a solid substrate to form supported membranes 13–16,19–21 for characterization purposes since the planar geometry of such supported membranes is compatible with many surface characterization tools 21–23 . However, some studies have shown that the outer leaflets of the cell membrane vesicles faced toward the bulk solution (that is, the outer leaflets were still facing outside-out) after they ruptured on the support 8–10,14,16 , whereas other studies have shown the opposite result – that is, that the inner leaflets faced toward the bulk solution 13,20 .…”

Creating Supported Plasma Membrane Bilayers Using Acoustic Pressure

“…In analogy to SLBs, there have also been efforts to create cell-derived (supported) planar model membrane systems. A common approach has so far been to disassemble cell-derived vesicles on glass surfaces by inducing the vesicle to burst with the help of additional artificial membrane patches [19][20][21], at air-water interfaces (thereby keeping the original cell membrane composition undisturbed) [22], or on polymer-supported lipid monolayers transferred from a Langmuir trough [23]. A common difficulty with all of these approaches is controlling which leaflet of the plasma membrane presents outwards (i.e., away from the support) which is essential when these systems are used to study molecular interactions.…”

Creating supported plasma membrane bilayers using acoustic pressure

“…In analogy to SLBs, there have consequently also been efforts to create cell-derived (supported) planar model membrane systems. A common approach has so far been to disassemble cell-derived vesicles on glass surfaces by inducing the vesicle to burst with the help of additional artificial membrane patches [19][20][21], at air-water interfaces (thereby keeping the original cell membrane composition undisturbed) [22], or on polymer-supported lipid monolayers transferred from a Langmuir trough [23]. A common difficulty with all of these approaches is controlling which leaflet of the plasma membrane presents outwards (i.e.…”