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. 2018 May 17;8(2):28.
doi: 10.3390/biom8020028.

Determination of the Membrane Environment of CD59 in Living Cells

Gergő Fülöp  1 , Mario Brameshuber  2 , Andreas M Arnold  3 , Gerhard J Schütz  4 , Eva Sevcsik  5
Affiliations

Determination of the Membrane Environment of CD59 in Living Cells

Gergő Fülöp et al. Biomolecules. .

Abstract

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid environment and vice versa. Studies on model membranes and plasma membrane vesicles indicated preferences of proteins for lipid phases characterized by different acyl chain order; however, whether such phases do indeed exist in live cells is still not known. Here, we refine a previously developed micropatterning approach combined with single molecule tracking to quantify the influence of the glycosylphosphatidylinositol-anchored (GPI-anchored) protein CD59 on its molecular environment directly in the live cell plasma membrane. We find that locally enriched and immobilized CD59 presents obstacles to the diffusion of fluorescently labeled lipids with a different phase-partitioning behavior independent of cell cholesterol levels and type of lipid. Our results give no evidence for either specific binding of the lipids to CD59 or the existence of nanoscopic ordered membrane regions associated with CD59.

Keywords: CD59; GPI-anchored protein; diffusion; lipid; membrane rafts; micropatterning; plasma membrane.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Principle of the experimental design and data analysis. (A) Antibody patterns are used to enrich and immobilize CD59-monomeric green fluorescent protein (mGFP) at specific sites in the plasma membrane of living T24 cells. Cells are interfaced with microstructured surfaces containing different densities of antibody, so that different surface densities of immobilized CD59-mGFP can be adjusted; (B) Typical region of interest in a micropatterned cell showing enrichment of CD59-mGFP in “ON” areas and depletion in “OFF” areas. Scale bar is 3 µm; (C) Sketch depicting individual immobilized CD59-mGFP molecules (dots) in “ON” areas. Trajectories of fluorescently labeled lipids (shown in red) are recorded and separated into the categories “ON” and “OFF” according to the CD59-mGFP patterns recorded in the GFP color channel.
Figure 2
Figure 2
Mobility of the cholesterol analog Chol-PEG-KK114 decreases with increasing CD59-mGFP surface density. (A) Mean square displacements (msd) of “ON” (orange) and “OFF” (black) CD59-mGFPI areas are plotted as a function of tlag for one representative cell. Diffusion coefficients were determined by fitting the function msd = 4Dtlag + 4σxy2; (B) Relative diffusion coefficients DON/DOFF were determined for individual cells (N = 22), plotted as a function of CD59-mGFP density, ρCD59-mGFP, and fitted with Equation (2) (steric obstacles, orange line) or Equation (1) assuming a combined size of tracer and obstacle d = 2 nm (binding, dotted grey line). The gray circle indicates the cell shown in (A).
Figure 3
Figure 3
Dioleoyl-phosphatidylethanolamine(DOPE)-PEG-KK114 diffusion decreases with increasing CD59-mGFP density; the relative mobility of both lipids is independent of cell cholesterol levels. (A) Mean square displacements of DOPE-PEG-KK114 “ON” (orange) and “OFF” (black) CD59-mGFPI-enriched areas are plotted as a function of tlag for one representative cell. Diffusion coefficients were determined by fitting the function msd = 4Dtlag + 4σxy2; (B) Relative diffusion coefficients DON/DOFF were determined for individual cells (N = 28), plotted as a function of CD59-mGFP density, ρCD59-mGFP, and fitted with Equation (2) (orange line). The gray circle indicates the cell shown in (A). Relative diffusion coefficients DON/DOFF are plotted as a function of CD59-mGFP density (open diamonds) and after treatment with 1 U/mL cholesterol oxidase (closed diamonds) for (C) DOPE-PEG-KK114 (N = 19) and (D) Chol-PEG-KK114 (N = 17). Data from cholesterol-depleted cells were fitted with Equation (2) (orange line).
Figure 4
Figure 4
Sketch of CD59-mGFP and DOPE-PEG-KK114. We have used information from [27,34,35,37,38] for realistic size estimates of the CD59 core protein, its GPI anchor, and the C-terminally attached mGFP, as well as the lipid, the PEG linker, and the fluorophore.
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