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 (HP1α) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) 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.
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We thank G. Patterson and J. Lippincott-Schwartz for kindly providing the PA-GFP–C1 vector, and R. Tsien for the mRFP1 cDNA. We thank M. Logsdon for technical assistance, Y. Chen from the Keck Center for Cellular Imaging for assistance with FLIM data analysis, and F. Koberling (PicoQuant GmbH) for helpful discussion. We also thank J. Redick and C. Davis from the Advanced Microscopy Facility for help with the laser scanning confocal microscopy. This work was supported by a grant from the US National Institutes of Health (DK47301 to R.N.D.).
Wide-field microscope images showing the nuclei of mouse GHFT1 cells that expressed either YFP-HP1α or YFP-C/EBPα. (PDF 535 kb)
The full blot from the co-immunoprecipitation analysis of the association of HP1α and C/EBPα. (PDF 156 kb)
Control experiments monitoring the photobleaching of CFP under conditions used for the photoactivation of PA-GFP. (PDF 405 kb)
The mean donor lifetime distributions obtained by two-component analysis of CFP fluorescence lifetime from cells expressing: CFP-C/EBPα; CFP-C/EBPα and PA-GFP-HP1α; CFP-C/EBP BZIP and PA-GFP-CEBP BZIP. (PDF 926 kb)
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Applications of fluorescence and bioluminescence resonance energy transfer to drug discovery at G protein coupled receptors
Analytical and Bioanalytical Chemistry (2010)