期刊
FASEB JOURNAL
卷 25, 期 9, 页码 2883-2897出版社
FEDERATION AMER SOC EXP BIOL
DOI: 10.1096/fj.11-181537
关键词
fluorescence microscopy; GPI-anchored proteins; ligand binding; receptor dimerization
资金
- Cariplo Foundation
- Italian Association for Cancer Research (AIRC)
- Spanish Ministry of Science and Innovation
- Pro-CNIC Foundation
- National Center for Research Resources of the U.S. National Institutes of Health [PHS 5 P41-RR003155]
- UCI
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.-Hellriegel, C., Caiolfa, V. R., Corti, V., Sidenius, N., Zamai, M. Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane. FASEB J. 25, 2883-2897 (2011). www.fasebj.org
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