DOI: 10.1210/mend.12.9.0168. Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy

Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy

10.1210/mend.12.9.0168

Crossref journal-article

The Endocrine Society

Molecular Endocrinology (80)

Abstract

AbstractThe pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 Å. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.

Bibliography

Day, R. N. (1998). Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy. Molecular Endocrinology, 12(9), 1410â1419.

Authors 1

Richard N. Day (first)

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Dates

Type	When
Created	11 years, 7 months ago (Jan. 8, 2014, 12:29 p.m.)
Deposited	3 years, 5 months ago (March 21, 2022, 8:21 p.m.)
Indexed	4 months ago (April 26, 2025, 3:23 a.m.)
Issued	26 years, 11 months ago (Sept. 1, 1998)
Published	26 years, 11 months ago (Sept. 1, 1998)
Published Print	26 years, 11 months ago (Sept. 1, 1998)

Funders 0

None

BibTeX

@article{Day_1998, title={Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy}, volume={12}, ISSN={1944-9917}, url={http://dx.doi.org/10.1210/mend.12.9.0168}, DOI={10.1210/mend.12.9.0168}, number={9}, journal={Molecular Endocrinology}, publisher={The Endocrine Society}, author={Day, Richard N.}, year={1998}, month=sep, pages={1410–1419} }

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