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

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.

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. 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, ρ_CD59,ON/ρ_CD59,OFF, is plotted as a function of the surface density of mGFP-GPI, ρ_mGFP-GPI, for 31 individual cells (six independent experiments).

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 t_lag for one representative cell. Diffusion coefficients were determined by fitting the function MSD = 4Dt_lag + 4σ²_xy (bold lines). Only the first two data points were considered for fits. We also included an anomalous diffusion fit with MSD = 4Dt_lagα + 4σ²_xy(hairlines), yielding α_ON=0.940 and α_OFF=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 D_ON/D_OFF were determined for 19 individual cells in four independent experiments and plotted as a function of (b) mGFP-GPI density ρ_mGFP-GPI or (c) average mGFP-GPI spacing d_mGFP-GPI. Data were fitted with D_ON/D_OFF = 1 - d₀²/d_mGFP-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 D_ON/D_OFF of Chol-KK114 as a function of average mGFP-GPI spacing d_mGFP-GPI in T24 cells (open diamonds) and in HeLa cells (closed diamonds, N=9, three independent experiments).

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 t_lag for a representative cell. Diffusion coefficients were determined by fitting the function MSD = 4Dt_lag + 4σ²_xy (bold lines). Only the first two data points were considered for fits. We also included an anomalous diffusion fit with MSD = 4Dt_lagα + 4σ²_xy (hairlines), yielding α_ON=0.917 and α_OFF=0.949. The first 10 datapoints were considered for fits. The inset shows a magnification of the first four data points. Relative diffusion coefficients D_ON/D_OFF of CD59 were determined for 31 individual cells in six independent experiments and plotted as a function of (b) mGFP-GPI density ρ_mGFP-GPI or (c) average mGFP-GPI spacing d_mGFP-GPI. Data were fitted with D_ON/D_OFF = 1 - d₀²/d_mGFP-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 D_ON/D_OFF of CD59 as a function of average mGFP-GPI spacing d_mGFP-GPI in T24 cells (open diamonds) and in HeLa cells (closed diamonds, N=11, three independent experiments).

Figure 5. Effect of cholesterol depletion and temperature change on CD59 mobility

(a) Relative diffusion coefficients D_ON/D_OFF of CD59 as a function of average mGFP-GPI spacing d_mGFP-GPI (open diamonds) and after treatment with 1 U/mL cholesterol oxidase (closed diamonds) (N=34, six independent experiments). (b) Relative diffusion coefficients D_ON/D_OFF of CD59 as a function of average mGFP-GPI spacing d_mGFP-GPI at 23°C (open diamonds) and at 37°C (closed diamonds) (N=11, three independent experiments).

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 δ_A→B. For the interaction pair mGFP-GPI (green) and CD59 (red), δ_{mGFP-GPI→CD59} is determined by the GFP moiety and the glycosylated exoplasmic part of CD59 yielding δ_{mGFP-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 δ_{mGFP-GPI→Chol-KK114} of 1.6 nm.