title: Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane.
abstract: We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.
authors: Microscopy and Dynamic Imaging Unit, National Center for Cardiovascular Research (CNIC), Madrid, Spain.
authors: Christian
authors: C
authors: Hellriegel
authors: Valeria R
authors: VR
authors: Caiolfa
authors: Valeria
authors: V
authors: Corti
authors: Nicolai
authors: N
authors: Sidenius
authors: Moreno
authors: M
authors: Zamai
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