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

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PROXIMAL MOLECULAR PROBE TRANSFER (PROMPT), A NEW APPROACH FOR IDENTIFYING SITES OF PROTEIN/NUCLEIC ACID INTERACTION IN CELLS BY CORRELATED LIGHT AND ELECTRON MICROSCOPY Article Open access

05 December 2023 TAGBIFC TECHNIQUE ALLOWS LONG-TERM SINGLE-MOLECULE TRACKING OF PROTEIN-PROTEIN INTERACTIONS IN LIVING CELLS Article Open access 19 March 2021 EXTENDING FLUORESCENCE

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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 AUTHORS AND AFFILIATIONS * Departments of Medicine and Cell Biology, P.O. Box 800578, University of Virginia Health Sciences Center, Charlottesville, 22908, Virginia, USA

Ignacio A Demarco, Cynthia F Booker & Richard N Day * W.M. Keck Center for Cellular Imaging, University of Virginia, Gilmer Hall, Charlottesville, 22904, Virginia, USA Ammasi Periasamy

Authors * Ignacio A Demarco View author publications You can also search for this author inPubMed Google Scholar * Ammasi Periasamy View author publications You can also search for this

author inPubMed Google Scholar * Cynthia F Booker View author publications You can also search for this author inPubMed Google Scholar * Richard N Day View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to Richard N Day. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing financial interests. 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) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Demarco, I., Periasamy, A., Booker, C. _et al._ Monitoring

dynamic protein interactions with photoquenching FRET. _Nat Methods_ 3, 519–524 (2006). https://doi.org/10.1038/nmeth889 Download citation * Received: 21 April 2006 * Accepted: 15 May 2006

* Published: 21 June 2006 * Issue Date: July 2006 * DOI: https://doi.org/10.1038/nmeth889 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get

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