title: Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.
abstract: Our understanding of complex biological systems is based on high-quality proteomics tools for the parallelized detection and quantification of protein interactions. Current screening platforms, however, rely on measuring protein interactions in rather artificial systems, rendering the results difficult to confer on the in vivo situation. We describe here a detailed protocol for the design and the construction of a system to detect and quantify interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in living cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput, making it applicable as a screening tool. The proof-of-concept is demonstrated for the interaction between CD4, a major coreceptor in T-cell signaling, and Lck, a protein tyrosine kinase essential for early T-cell signaling.
authors.affiliation: Biophysics Institute, Johannes Kepler University Linz, Linz, Austria.
authors.forename: Julian
authors.initials: J
authors.lastname: Weghuber
authors.forename: Mario
authors.initials: M
authors.lastname: Brameshuber
authors.forename: Stefan
authors.initials: S
authors.lastname: Sunzenauer
authors.forename: Manuela
authors.initials: M
authors.lastname: Lehner
authors.forename: Christian
authors.initials: C
authors.lastname: Paar
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselgrübler
authors.forename: Michaela
authors.initials: M
authors.lastname: Schwarzenbacher
authors.forename: Martin
authors.initials: M
authors.lastname: Kaltenbrunner
authors.forename: Clemens
authors.initials: C
authors.lastname: Hesch
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Paster
authors.forename: Bettina
authors.initials: B
authors.lastname: Heise
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
authors.forename: Hannes
authors.initials: H
authors.lastname: Stockinger
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods; D013499:Surface Properties
chemical_list: D015704:CD4 Antigens; D005456:Fluorescent Dyes; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)