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. 2015 Apr 21:6:6969.
doi: 10.1038/ncomms7969.

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

Eva Sevcsik  1 , Mario Brameshuber  1 , Martin Fölser  1 , Julian Weghuber  2 , Alf Honigmann  3 , Gerhard J Schütz  1
Affiliations

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

Eva Sevcsik et al. Nat Commun. .

Abstract

The organization of proteins and lipids in the plasma membrane has been the 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.

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Figures

Figure 1
Figure 1. mGFP-GPI patterning scheme
(a) Cells stably transfected with mGFP-GPI were grown on micropatterned glass coverslips containing 3 μm sized spots of GFP antibody; interspaces were passivated by BSA. mGFP-GPI became locally immobilized and enriched at plasma membrane regions coinciding with the GFP antibody patterns. (b) Photoactivated localization microscopy (PALM) image of psCFP-2 bound to anti-GFP patterns. Scale bar is 1 μm. (c) Total internal reflection microscopy (TIRFM) image of a cell grown on micropatterns. mGFP-GPI clearly follows the antibody patterns. The cell outline is indicated by the dashed white contour line. Scale bar is 10 μm. (d) Schematic of our approach. Cells are grown on slides with micropatterns containing different amounts of GFP antibody, so that different surface densities of immobilized mGFP-GPI, ρ, can be adjusted in the plasma membrane. Localizations and mobilities of a probe (CD59 or Chol-KK114) are determined for mGFP-GPI enriched (ON) and depleted (OFF) areas.
Figure 2
Figure 2. CD59 is not recruited to mGFP-GPI areas
(a) The positions of single Fab-Atto 647N labeled CD59 molecules were recorded within a selected region of interest; no apparent correlation with the underlying mGFP-GPI patterns (red circles) is visible. Scale bar is 3 μm. (b) The relative surface density of CD59, ρCD59,ON/ρCD59,OFF, is plotted as a function of the surface density of mGFP-GPI, ρmGFP-GPI, for 31 individual cells (six independent experiments).
Figure 3
Figure 3. Tracer mobility is only minimally affected by mGFP-GPI enrichment
(a) Mean square displacements of Chol-KK114 ON (black) and OFF (grey) mGFP-GPI enriched areas are plotted as a function of tlag for one representative cell. Diffusion coefficients were determined by fitting the function MSD = 4Dtlag + 4σ2xy (bold lines). Only the first two data points were considered for fits. We also included an anomalous diffusion fit with MSD = 4Dtlagα + 4σ2xy(hairlines), yielding αON=0.940 and αOFF=0.922. The first 10 datapoints were considered for fits. The inset shows a magnification of the first four data points. Only minor deviation from Brownian motion was observed ON as well as OFF mGFP-GPI areas. Relative diffusion coefficients DON/DOFF were determined for 19 individual cells in four independent experiments and plotted as a function of (b) mGFP-GPI density ρmGFP-GPI or (c) average mGFP-GPI spacing dmGFP-GPI. Data were fitted with DON/DOFF = 1 - d02/dmGFP-GPI2 (c) yielding the characteristic nearest-neighbor distance of mGFP-GPI at the percolation threshold d0 = 2.6±0.5 nm. The gray circle indicates the ratio of diffusion coefficients shown in (a). The grey lines represent the standard error of the fit. (d) Relative diffusion coefficients DON/DOFF of Chol-KK114 as a function of average mGFP-GPI spacing dmGFP-GPI in T24 cells (open diamonds) and in HeLa cells (closed diamonds, N=9, three independent experiments).
Figure 4
Figure 4. Mobility of CD59 decreases linearly with increasing mGFP-GPI surface density
(a) Mean square displacements of CD59 ON (black) and OFF (grey) mGFP-GPI enriched areas as a function of tlag for a representative cell. Diffusion coefficients were determined by fitting the function MSD = 4Dtlag + 4σ2xy (bold lines). Only the first two data points were considered for fits. We also included an anomalous diffusion fit with MSD = 4Dtlagα + 4σ2xy (hairlines), yielding αON=0.917 and αOFF=0.949. The first 10 datapoints were considered for fits. The inset shows a magnification of the first four data points. Relative diffusion coefficients DON/DOFF of CD59 were determined for 31 individual cells in six independent experiments and plotted as a function of (b) mGFP-GPI density ρmGFP-GPI or (c) average mGFP-GPI spacing dmGFP-GPI. Data were fitted with DON/DOFF = 1 - d02/dmGFP-GPI2 yielding the characteristic nearest-neighbor distance of mGFP-GPI at the percolation threshold d0 = 6.3±0.2 nm. The gray circle indicates the data point shown in (a). The grey lines represent the standard error of the fit. (d) Relative diffusion coefficients DON/DOFF of CD59 as a function of average mGFP-GPI spacing dmGFP-GPI in T24 cells (open diamonds) and in HeLa cells (closed diamonds, N=11, three independent experiments).
Figure 5
Figure 5. Effect of cholesterol depletion and temperature change on CD59 mobility
(a) Relative diffusion coefficients DON/DOFF of CD59 as a function of average mGFP-GPI spacing dmGFP-GPI (open diamonds) and after treatment with 1 U/mL cholesterol oxidase (closed diamonds) (N=34, six independent experiments). (b) Relative diffusion coefficients DON/DOFF of CD59 as a function of average mGFP-GPI spacing dmGFP-GPI at 23°C (open diamonds) and at 37°C (closed diamonds) (N=11, three independent experiments).
Figure 6
Figure 6. Tracers experience varying degrees of friction at different distances from the membrane surface
The reach of a membrane-associated molecule A with respect to a second molecule B can be defined as its repulsion diameter δA→B. For the interaction pair mGFP-GPI (green) and CD59 (red), δmGFP-GPI→CD59 is determined by the GFP moiety and the glycosylated exoplasmic part of CD59 yielding δmGFP-GPI→CD59= 2.5 nm. In contrast, Chol-KK114 (blue) can approach the acyl chains of the GPI-anchor, giving rise to a repulsion diameter δmGFP-GPI→Chol-KK114 of 1.6 nm.
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