PMID- 21602447
OWN - NLM
STAT- MEDLINE
DCOM- 20111109
LR  - 20211020
IS  - 1530-6860 (Electronic)
IS  - 0892-6638 (Print)
IS  - 0892-6638 (Linking)
VI  - 25
IP  - 9
DP  - 2011 Sep
TI  - Number and brightness image analysis reveals ATF-induced dimerization kinetics of 
      uPAR in the cell membrane.
PG  - 2883-97
LID - 10.1096/fj.11-181537 [doi]
AB  - 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.
FAU - Hellriegel, Christian
AU  - Hellriegel C
AD  - Microscopy and Dynamic Imaging Unit, National Center for Cardiovascular Research 
      (CNIC), Madrid, Spain.
FAU - Caiolfa, Valeria R
AU  - Caiolfa VR
FAU - Corti, Valeria
AU  - Corti V
FAU - Sidenius, Nicolai
AU  - Sidenius N
FAU - Zamai, Moreno
AU  - Zamai M
LA  - eng
GR  - P41 RR003155/RR/NCRR NIH HHS/United States
GR  - 5 P41-RR003155/RR/NCRR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20110520
PL  - United States
TA  - FASEB J
JT  - FASEB journal : official publication of the Federation of American Societies for 
      Experimental Biology
JID - 8804484
RN  - 0 (Receptors, Urokinase Plasminogen Activator)
RN  - 0 (Vitronectin)
RN  - 0 (enhanced green fluorescent protein)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - Algorithms
MH  - Cell Membrane/*metabolism
MH  - Computer Simulation
MH  - Gene Expression Regulation/physiology
MH  - Green Fluorescent Proteins/metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - *Image Processing, Computer-Assisted
MH  - Immunoblotting
MH  - Microscopy, Fluorescence/instrumentation/*methods
MH  - Protein Multimerization
MH  - Receptors, Urokinase Plasminogen Activator/*metabolism
MH  - Reproducibility of Results
MH  - Vitronectin/metabolism
PMC - PMC3157682
EDAT- 2011/05/24 06:00
MHDA- 2011/11/10 06:00
PMCR- 2012/09/01
CRDT- 2011/05/24 06:00
PHST- 2011/05/24 06:00 [entrez]
PHST- 2011/05/24 06:00 [pubmed]
PHST- 2011/11/10 06:00 [medline]
PHST- 2012/09/01 00:00 [pmc-release]
AID - fj.11-181537 [pii]
AID - 11-181537 [pii]
AID - 10.1096/fj.11-181537 [doi]
PST - ppublish
SO  - FASEB J. 2011 Sep;25(9):2883-97. doi: 10.1096/fj.11-181537. Epub 2011 May 20.