Micropatterning of Plasma Membrane Proteins to Analyze Raft Localization in Living Cells

Abstract

We have developed an assay for quantitative analysis of the interaction between a fluorescently marked protein (prey) and a membrane protein (bait) using microstructured surfaces covered with biotinylated ligands (antibodies) targeted against the bait. The proof-of-concept was demonstrated for the interaction between CD4, a major co-receptor in T-cell signalling, and Lck, a protein tyrosine kinase essential for early T cell signalling. Here we present improvements and a more precise characterization of the method as well as the applicability of the assay for the analysis of protein interactions within lipid rafts in the inner and outer leaflet of the plasma membrane. We stably expressed fluorescently labelled raft and non-raft proteins in the human T24 cell line as prey proteins and determined the degree of interaction with the antibody-targeted bait proteins CD59 (GPI-anchored protein, raft marker) and CD71 (Transferrin-receptor, non-raft marker), respectively. We found strong interaction of CD59 with putative raft markers including various GPI-GFP constructs, the inner-leaflet associated proteins Lck and Flottilin1 and a Pleckstrin-Homology domain fused to GFP. Importantly, we did not find interaction of CD59 with CD71-GFP and other potential non-raft proteins. When CD71 was used as the bait protein we did not find interaction with the putative raft markers. While the detected absence of CD71 from and the presence of CD59 in lipid rafts confirm current knowledge, it is still very unclear if a lipid-raft dependent coupling of proteins and certain especially negatively charged lipids across the plasma-membrane bilayer exists. Thus, our micropatterning assay will be of great interest to address this question.