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.
Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46, 02-668, Warsaw, Poland.
Remigiusz
R
Worch
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 10-12/I, 8010, Graz, Austria.
Zdenek
Z
Petrášek
Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany.
Petra
P
Schwille
Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany. weidemann@biochem.mpg.de.
Thomas
0000-0002-6694-1087
T
Weidemann
D002232:Carbocyanines / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism*; D053662:Interleukin-4 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
C569686:Alexa Fluor 647; D002232:Carbocyanines; D005456:Fluorescent Dyes; C471213:His-His-His-His-His-His; C508598:IL4 protein, human; C504000:IL4R protein, human; D053662:Interleukin-4 Receptor alpha Subunit; D009842:Oligopeptides; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins; D053835:Unilamellar Liposomes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015847:Interleukin-4; D006639:Histidine; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine
Diffusion coefficient; Fluorescence correlation spectroscopy; Fluorescent lipid; Giant plasma membrane vesicles; Giant unilamellar vesicles; Interleukin-4 receptor