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.
Highlights
The nucleus comprises different subdomains whose biological functions remain often very elusive, if known at all, and that differ fundamentally from cytoplasmic compartments in that they are not delineated by membranes [1, 2]
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
Characterization of enhanced yellow fluorescent protein (EYFP)-PPAR Chimeras—To monitor PPAR action in living cells, we constructed expression vectors for the different PPAR isotypes fused to EYFP either at their N or C terminus (EYFP-PPAR and PPAR-EYFP, respectively)
Summary
Materials—Rosiglitazone and 17-estradiol were purchased from Sigma, Wy14,643 from Cayman Chemical Co. (Ann Arbor, MI), and 9-cis-retinoic acid from Biomol Laboratories (Plymouth Meeting, PA). The antibody directed against PPAR␣ has been previously described [33], and those directed against PPAR, PPAR␥, and GFP were purchased from Affinity Bioreagents (Golden, CO), Wak-Chemie (Steinbach, Germany), and Roche Diagnostics (Rotkreuz, Switzerland), respectively. For cofactor pull-downs, the GSTp3002–516 fusion protein was purified as described previously [34] For both DNA and cofactor pull-downs, whole-cell extracts from transfected COS-7 cells were incubated 2.5 h at 4 °C in pull-down buffer in the oligonucleotide coated plates or with the GST-p3002–516 coated beads, and the appropriate PPAR ligands (Wy14,643 at 100 M, L-165041 at 50 M, and rosiglitazone at 10 M). Antibody incubations were followed by two 5-min washes in PBS, 2% BSA, and samples were mounted in DAPI containing-Vectashield mounting medium (Vector Laboratories). Fluorescence intensities were calculated in the bleached zone and over the entire cell nucleus with NIH ImageJ version 1.30 or Zeiss LSM softwares. The sizes of the EYFP-PPAR complexes were estimated by multiplying the molecular mass of EYFP by the factor (DEYFP/DEYFP-PPAR), assuming spherical symmetry of the complexes and normal diffusion in the viscous medium of the nucleus (i.e. EYFP experiences the same viscosity as the EYFP-PPAR complexes [40])
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