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Articles by Stefan Sunzenauer in JoVE
JoVE General
In vivo Detektion av protein-protein interaktioner på mikro-mönstrade ytor
Institute of Biophysics, Johannes Kepler Universitat Linz
Denna video visar experiment med efterföljande analys av protein-protein interaktioner med hjälp av mikro-mönstrade ytor. Den metod som ger möjlighet att upptäcka protein interaktioner i levande celler och kombinerar hög genomströmning kapacitet med möjlighet att utvinna kvantitativ information.
Other articles by Stefan Sunzenauer on PubMed
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.
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.
VEGF-INDUCED ENDOTHELIAL CELL MIGRATION REQUIRES UROKINASE RECEPTOR (uPAR)-DEPENDENT INTEGRIN REDISTRIBUTION
AIMS: Vascular endothelial growth factor (VEGF)-initiated angiogenesis requires coordinated proteolytic degradation of extracellular matrix provided by the urokinase plasminogen activator/urokinase receptor (uPA/uPAR) system and regulation of cell-migration provided by integrin-matrix interaction. In this study we investigated the mechanisms underlying the uPAR-dependent modulation of VEGF-induced endothelial migration.Methods and ResultsWe used flow cytometry to quantify integrins at the cell surface. Stimulation of human and murine endothelial cells with VEGF resulted in internalization of α5β1-integrins. Micropatterning and immuno-cytochemistry revealed co-clustering of uPAR and α5β1-integrins and retrieval via clathrin-coated vesicles. It was also contingent on receptors of the low density lipoprotein receptor (LDL-R) family. VEGF-induced integrin redistribution was inhibited by elimination of uPAR from the endothelial cell surface or by inhibitory peptides that block the uPAR/integrin interaction. Under these conditions the migratory response of endothelial cells upon VEGF stimulation was impaired both, in vitro and in vivo. CONCLUSIONS: The observations indicate that uPAR is an essential component of the network through which VEGF controls endothelial cell migration. UPAR is a bottleneck through which the VEGF-induced signal must be funnelled for both, focused proteolytic activity at the leading edge and for redistribution of integrins.
