Temporal resolution of protein-protein interactions in the live-cell plasma membrane.
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.
Weghuber
Julian
J
Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr.69, 4040 Linz, Austria.
Sunzenauer
Stefan
S
Plochberger
Birgit
B
Brameshuber
Mario
M
Haselgrübler
Thomas
T
Schütz
Gerhard J
GJ
D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002477:Cells / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D040081:Protein Array Analysis; D011485:Protein Binding
D008565:Membrane Proteins