Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.
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.
Weghuber
Julian
J
Biophysics Institute, Johannes Kepler University Linz, Linz, Austria.
Brameshuber
Mario
M
Sunzenauer
Stefan
S
Lehner
Manuela
M
Paar
Christian
C
Haselgrübler
Thomas
T
Schwarzenbacher
Michaela
M
Kaltenbrunner
Martin
M
Hesch
Clemens
C
Paster
Wolfgang
W
Heise
Bettina
B
Sonnleitner
Alois
A
Stockinger
Hannes
H
Schütz
Gerhard J
GJ
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
D015704:CD4 Antigens; D005456:Fluorescent Dyes; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)