Measuring uPAR Dynamics in Live Cells
Urokinase-type plasminogen activator receptor (uPAR) is a key component of the urokinase plasminogen activation (uPA) system, which plays important roles in physiological processes as well as in tumor invasion and metastasis. Besides uPAR’s well-established role in the regulation of pericellular proteolysis, a large body of evidences suggests that several of uPAR-mediated events do not require the proteolytic activity of uPA. The common accepted notion is that uPAR transduces signals through direct lateral physical interactions in multimolecular complexes involving membrane-spanning proteins and extracellular surface proteins. However, none of these interactions have ever been visualized and confirmed in living cells, at steady state and in the absence of crosslinkers or antibody clustering agent. Thus, the physical engagement of uPAR in its monomeric or oligomeric forms in multimolecular complexes needs to be convincingly verified observing uPAR at work. This chapter discusses how fully functional fluorescent protein-tagged uPAR chimeras can be generated and used for determining distribution, recruitment, mobility, monomer-dimer exchange, and biologically relevant uPAR interactions in living cells, and in unperturbed conditions.
KeywordsHEK293 Cell Forster Resonance Energy Transfer Fluorescence Correlation Spectroscopy Urokinase Receptor Fluorescence Lifetime Imaging Microscopy
Unable to display preview. Download preview PDF.