A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

Abstract

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.

Fig. 1.

Size gating of liposomes in FACS sideward to forward scatter. “Deleted events” corresponds to filtered background noise. The 0.1 μm (black), 0.3 μm (red), 0.6 μm (yellow), and 1.1 μm (blue) boxes correspond to size gates defined by measurement of beads with known sizes. Both axes are in logarithmic scale. A: Merge of the 100% linear density plots of 0.1 μm, 0.3 μm, 0.6 μm, and 1.1 μm latex beads. B: Density plot (100% linear) of liposomes generated by size extrusion. C: Dot plot corresponding to the liposomes in B, showing a broad size range. D: Average size distribution of liposomes (n = 6) as obtained by dynamic light scatter.

Fig. 2.

Rhodamine-based gating of liposomes. A: Histogram overlay of liposomes without (black) and with 1 mol% rhod-PE (red), defining a cut-off value for rhodamine-positive liposomes where <1% false positives are detected. B: Dot plot of forward scatter to rhodamine-derived fluorescence showing the rhodamine gate (gated events) and the size gates as described in the legend to Fig. 1. Axes are in logarithmic scale.

Fig. 3.

Detection of liposomes with bound GFP fusion proteins using flow cytometry. A: Dot plot of forward scatter to GFP-derived fluorescence, showing liposomes with 5 mol% PI(4,5)P₂ treated with 5 μM FGF-2-GFP. Gating parameters are as described in Fig. 2B. Axes are in logarithmic scale. B: Histogram of FGF-2-GFP binding to liposomes with 5 mol% PI(4,5)P₂ having a 0.6 μm diameter. The GFP-derived signal distribution of the 30,000 measured events as well as the population geo mean are depicted. Data were filtered through gates as described in Fig. 2B. The x axis is in logarithmic scale. C: Titration curve of liposomes with varying PI(4,5)P₂ content treated with 5 μM FGF-2-GFP. Curves represent he different size gates defined in Fig. 2B. The c.F.U. values (y axis, log scale) are obtained by subtracting the geo mean of the background sample (GFP-treated) from the geo mean of the FGF-2-GFP-treated sample and dividing the outcome by a shape index, a correction factor for liposome aggregation and deformation. D, E: Standard curves for the conversion of GFP-derived F.U. to molecules of GFP (D) and of rhodamine-derived F.U. to molecules of PI(4,5)P₂ (E). Axes depict the logarithmic values (see “data processing”). Equations and graphs represent the average of three independent experiments and the R² exceed 0.99.

Fig. 4.

Titration curves of binding to liposomes of PH-PLCδ₁ and FGF-2. A, C: Linear fitting (full line) of the initial binding slope to liposomes dependent on liposome size gating for PH-PLCδ₁ (A) and FGF-2 (C). B, D: Nonlinear regression fit (full line) for binding of PH-PLCδ₁ (B) and FGF-2 (D) to liposomes for each liposome size gate. The dotted line represents the 95% confidence band. c.F.U. values are derived as described under “data processing.”

Fig. 5.

Presumed alterations of liposome shape upon protein binding. Liposomes were incubated with the proteins indicated and measured by flow cytometry using the gates defined in Fig. 2B. Shape index values (y axis) above 1 are indicative of an increase (negative membrane curvature or tethering), and values below 1 indicate a decrease (positive curvature) in rhodamine-derived signal averaged for all liposome size gates. Mean values of three independent experiments with standard deviations are shown.