title: Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand.
abstract: In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.
authors.affiliation: Center for Integrative Genomics, NCCR Frontiers in Genetics, University of Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Jérôme N
authors.initials: JN
authors.lastname: Feige
authors.forename: Laurent
authors.initials: L
authors.lastname: Gelman
authors.forename: Cicerone
authors.initials: C
authors.lastname: Tudor
authors.forename: Yves
authors.initials: Y
authors.lastname: Engelborghs
authors.forename: Walter
authors.initials: W
authors.lastname: Wahli
authors.forename: Béatrice
authors.initials: B
authors.lastname: Desvergne
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008024:Ligands; D047492:Peroxisome Proliferator-Activated Receptors / Q000032:analysis* / Q000378:metabolism*; D047488:Retinoid X Receptors / Q000032:analysis* / Q000378:metabolism*
chemical_list: D008024:Ligands; D047492:Peroxisome Proliferator-Activated Receptors; D047488:Retinoid X Receptors