June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Observation of CRX and NRL interactions in live single mammalian cells using laser scanning confocal microscopy and flow cytometry FRET
Author Affiliations & Notes
  • Barry E Knox
    Ophthalmology & Visual Sciences and Biochemistry & Mol Bio., SUNY Upstate Medical University, Syracuse, New York, United States
  • Xinming Zhuo
    Ophthalmology & Visual Sciences and Biochemistry & Mol Bio., SUNY Upstate Medical University, Syracuse, New York, United States
  • Footnotes
    Commercial Relationships   Barry Knox, None; Xinming Zhuo, None
  • Footnotes
    Support  NIH EY-11256 and EY-12975, Research to Prevent Blindness and the Lions of CNY.
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3781. doi:
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    • Get Citation

      Barry E Knox, Xinming Zhuo; Observation of CRX and NRL interactions in live single mammalian cells using laser scanning confocal microscopy and flow cytometry FRET. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3781.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : CRX and NRL are retina-specific transcription factors that together control photoreceptor differentiation, regulate gene expression, and when mutated underlie human retinopathies. Biochemical evidence has shown that CRX and NRL can bind to each other in vitro and cooperatively activate rod-specific gene promoters in cultured cells. Their interaction in the nuclear environment has not been previously characterized. Methods to observed fluorescently tagged transcription factors were developed for this purpose.

Methods : We examined their interaction in live mammalian cells (HEK293S) using fluorescence resonance energy transfer (FRET) through both laser scanning confocal microscopy and an improved flow cytometry method. FRET was measured using a ratiometric sensitized emission method, calibrated with a series of mCherry-eGFP (mG) fusion proteins. FRET efficiencies were measured over a wide-range of expression levels in transiently transfected cells.

Results : FRET efficiencies for CRX donor-acceptors were highest when donor and acceptor fluorophores were both fused to the N-termini, adjacent to the homeodomain, and were lower but detectable when were both fused to the C-terminal activation domains. FRET efficiencies for NRL donor-acceptor fusion proteins were highest when fluorophores were fused to the C-termini, adjacent to the basic leucine zipper (bZIP) domain and were lower when fluorophores were fused to the N-terminal activation domains. The FRET efficiencies for the CRX and NRL donor-acceptors were highest when fluorophores were fused to the CRX N-terminus and NRL C-terminus (near the bZip domain), respectively.

Conclusions : These results demonstrate the formation of CRX-NRL complexes in vivo in live cells, indicate their DNA binding regions are spatially close for a significant proportion of molecules, and opens the possibility of flow-cytometry based FRET for structure-function studies.

This is a 2020 ARVO Annual Meeting abstract.

 

Orientation of CRX and NRL complexes as determined by FRET measurements

Orientation of CRX and NRL complexes as determined by FRET measurements

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