Diffusion of Single-Pass Transmembrane Receptors: From the Plasma Membrane into Giant Liposomes.
To quantitatively examine the effect of membrane organization on lateral diffusion, we studied fluorescent carbocyanine lipid analogues and EGFP-tagged, single-pass transmembrane proteins in systems of decreasing complexity: (i) the plasma membrane (PM) of living cells, (ii) paraformaldehyde/dithiothreitol-induced giant plasma membrane vesicles (GPMVs), and (iii) giant unilamellar vesicles (GUVs) under physiological buffer conditions. A truncated, signaling-deficient interleukin-4 receptor subunit, showing efficient accumulation in the plasma membrane, served as a model transmembrane protein. Two-dimensional diffusion coefficients (D) were determined by fluorescence correlation spectroscopy (FCS) either at fixed positions (single-point, spFCS) or while scanning a circular orbit (circular scanning, csFCS). Consistent with a different inclusion sizes in the membrane, lipids diffuse slightly faster than the single-spanning membrane proteins in both membrane systems, GUVs and GPMVs. In GPMVs lipids and proteins consistently experienced a fivefold larger viscosity than in GUVs, reflecting the significant fraction of plasma membrane-derived proteins partitioning into GPMVs. Lipid and protein diffusion in the PM was, respectively, 2 times and 4-5 times slower in comparison to GPMVs. This discrepancy was quantitatively confirmed by csFCS. The similarity of diffusion of receptors and lipids in GPMVs and GUVs and its significant difference in the plasma membrane suggest that protein domains as small as EGFP convey sensitivity to the actin cortex on various length scales.
['Worch|Remigiusz|R|', 'Petrášek|Zdenek|Z|', 'Schwille|Petra|P|', 'Weidemann|Thomas|T|0000-0002-6694-1087']
D002232:Carbocyanines D002462:Cell Membrane D002784:Cholesterol D004058:Diffusion D005456:Fluorescent Dyes D015870:Gene Expression D049452:Green Fluorescent Proteins D057809:HEK293 Cells D006639:Histidine D006801:Humans D015847:Interleukin-4 D053662:Interleukin-4 Receptor alpha Subunit D007700:Kinetics D009842:Oligopeptides D010713:Phosphatidylcholines D010957:Plasmids D021381:Protein Transport D011993:Recombinant Fusion Proteins D013050:Spectrometry, Fluorescence D013109:Sphingomyelins D053835:Unilamellar Liposomes
Giant plasma membrane vesicles Giant unilamellar vesicles Interleukin-4 receptor Diffusion coefficient Fluorescence correlation spectroscopy Fluorescent lipid