Protein Micropatterning In Live Cells: A Tool For Creating Membrane Domains With Raft-Like Properties

Microstructured surfaces provide a unique and versatile platform to study cellular processes associated with the plasma membrane. In a recent study we presented a micropatterning assay to monitor protein-protein interactions in the live cell plasma membrane and used it to characterize the interaction between CD4, a major co-receptor in T cell signalling, and Lck, a protein tyrosine kinase essential for early T cell signaling (Schwarzenbacher, et al., Nat Methods, 2008). For this assay, cells are plated onto microstructured surfaces partly covered with ligands (antibodies) targeted against membrane proteins (bait), and the co-localization with a fluorescently labeled protein of interest (prey) is monitored.Here, we employ this technique to probe indirect protein-protein interactions, namely of the lipid raft-associated GPI(glycosylphosphatidylinositol)-anchored protein CD59 and a GPI-anchored GFP. Interestingly, antibody-mediated micropatterning of CD59 leads to a colocalization of GPI-GFP (and vice versa). The mechanism of this can be envisioned as follows: By patterning of one GPI-anchored protein, a certain membrane microenvironment is created and the other GPI-anchored protein preferentially localizes into this environment. We employ different compounds that have been reported to act as ‘raft’ or ‘non-raft’ markers to further characterize the nature of the generated membrane patterns.