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Number and brightness image analysis reveals ATF‐induced dimerization kinetics of uPAR in the cell membrane
Author(s) -
Hellriegel Christian,
Caiolfa Valeria R.,
Corti Valeria,
Sidenius Nicolai,
Zamai Moreno
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.11-181537
Subject(s) - urokinase receptor , hek 293 cells , chemistry , pentamer , photobleaching , biophysics , plasminogen activator , kinetics , receptor , activator (genetics) , biology , biochemistry , physics , fluorescence , genetics , optics , quantum mechanics
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