Monitoring dynamic protein interactions with photoquenching FRET.
The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy. We used this new method, photoquenching FRET (PQ-FRET), to define the dynamic interactions of the heterochromatin protein-1 alpha (HP1alpha) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) in regions of centromeric heterochromatin in mouse pituitary cells. The advantage of the PQ-FRET assay is that it provides simultaneous measurement of a protein's mobility, its exchange within macromolecular complexes and its interactions with other proteins in the living cell without the need for corrections based on reference images acquired from control cells.
Demarco
Ignacio A
IA
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Periasamy
Ammasi
A
Booker
Cynthia F
CF
Day
Richard N
RN
D000818:Animals; D015374:Biosensing Techniques; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism; D002460:Cell Line; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006570:Heterochromatin / Q000378:metabolism; D006801:Humans; D051379:Mice; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
C000715594:CBX5 protein, human; D022763:CCAAT-Enhancer-Binding Protein-alpha; D002868:Chromosomal Proteins, Non-Histone; D006570:Heterochromatin; D011506:Proteins; D000090266:Chromobox Protein Homolog 5