TY - JOUR
T1 - Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors
AU - Rehó, Bálint
AU - Lau, Lukas
AU - Mocsár, Gábor
AU - Müller, Gabriele
AU - Fadel, Lina
AU - Brázda, Péter
AU - Nagy, László
AU - Tóth, Katalin
AU - Vámosi, György
N1 - Publisher Copyright:
© 2019 American Chemical Society.
PY - 2020/1/21
Y1 - 2020/1/21
N2 - Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.
AB - Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.
UR - http://www.scopus.com/inward/record.url?scp=85078380578&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.9b04902
DO - 10.1021/acs.analchem.9b04902
M3 - Article
C2 - 31870146
AN - SCOPUS:85078380578
SN - 0003-2700
VL - 92
SP - 2207
EP - 2215
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 2
ER -