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Monitoring dynamic protein interactions with photoquenching FRET

Abstract

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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Acknowledgements

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.).

Author information

I.A.D. and R.N.D. contributed equally to the conceptual development of and implementation of this method. C.F.B. contributed to all technical aspects of this project. A.P. developed and helped apply the fluorescence lifetime measurements.

Competing interests

The authors declare no competing financial interests.

Correspondence to Richard N Day.

Supplementary information

Supplementary Fig. 1

Wide-field microscope images showing the nuclei of mouse GHFT1 cells that expressed either YFP-HP1α or YFP-C/EBPα. (PDF 535 kb)

Supplementary Fig. 2

The full blot from the co-immunoprecipitation analysis of the association of HP1α and C/EBPα. (PDF 156 kb)

Supplementary Fig. 3

Control experiments monitoring the photobleaching of CFP under conditions used for the photoactivation of PA-GFP. (PDF 405 kb)

Supplementary Fig. 4

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)

Supplementary Methods (DOC 29 kb)

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Further reading

Figure 1: Protein mobility and interactions can be measured using PA-GFP and PQ-FRET.
Figure 2: HP1α and C/EBPα interact in the nucleus of GHFT1 cells.
Figure 3: The mobility of HP1α within the nuclear compartment measured by PA-GFP and FRAP.
Figure 4: PQ-FRET is used to measure the dynamic interactions between HP1α and C/EBPα.
Figure 5: PQ-FRET is used to measure dimer formation by the C/EBP BZIP domains.