Abstract
The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.
Highlights
The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate
A decade later, Lo/Ld phase separation was demonstrated in giant plasma membrane vesicles (GPMVs) that can be generated from the plasma membrane of cells after specific treatment[14]
Immobilizing mGFP-GPI within patterns in the plasma membrane. mGFP-GPI is known to preferentially partition into the ordered phase of phase-separated GPMVs15, was found in detergent-resistant membrane (DRM) fractions[16] and is typically regarded as a bona fide raft marker
Summary
The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. We use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment This treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. Organization and interaction of proteins and lipids have been proposed to occur on different time and length scales, from direct molecular interactions to transient association within nanoscopic domains to the optically resolvable phase separation observed in model systems It is astonishing, that very basic functionalities of the plasma membrane have far remained enigmatic[2]. The underlying mechanisms for all these observations, are largely unclear; there are no experiments showing a direct link between the live cell data and fundamental membrane biophysics
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