Temporal resolution of protein-protein interactions in the live-cell plasma membrane

• Published in: Analytical and bioanalytical chemistry Vol. 397; no. 8; pp. 3339 - 3347
• Main Authors: Weghuber, Julian, Sunzenauer, Stefan, Plochberger, Birgit, Brameshuber, Mario, Haselgrübler, Thomas, Schütz, Gerhard J
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.08.2010; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Affinity labeling; Analysis; Analytical Chemistry; Antibodies; Atomic force microscopy; Baits; Bioanalysis; Biochemistry; Cell Line; Cell Membrane; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell membranes; Cells; Cells - chemistry; Cells - metabolism; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Contact; Fluctuation; Fluorescence; Fluorescence microscopy; Food Science; Green fluorescent protein; Humans; Kinetics; Laboratory Medicine; Laser scanning microscopy; Lipid rafts; Mathematical analysis; Medical research; Membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membranes; metabolism; Methods; Micro-patterned surfaces; Micropatterning; Microscopy; Monitoring; Monitoring/Environmental Analysis; Monitors; Morphology; Nucleation; Original Paper; Plasma; plasma membrane; Prey; Protein Array Analysis; Protein Binding; Protein interaction; Protein microarrays; Protein seeding; Protein-protein interactions; Proteins; sowing; Temporal resolution; Topography; Austria; Atomic force microscopy; Micro-patterned surfaces; Protein–protein interactions; Plasma membrane; Temporal resolution; Lipid rafts; Fluorescence microscopy
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-010-3854-x

We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053-1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section.