Interaction of human CRX and NRL in live HEK293T cells measured using fluorescence resonance energy transfer (FRET).
Sci Rep
; 12(1): 6937, 2022 04 28.
Article
en En
| MEDLINE
| ID: mdl-35484285
ABSTRACT
CRX and NRL are retina-specific transcription factors that control rod photoreceptor differentiation and synergistically activate rod phototransduction gene expression. Previous experiments showed they interact in vitro and in yeast two-hybrid assays. Here, we examined CRX-NRL interaction in live HEK293T cells using two fluorescence resonance energy transfer (FRET) approaches confocal microscopy and flow cytometry (FC-FRET). FC-FRET can provide measurements from many cells having wide donor-acceptor expression ranges. FRET efficiencies were calibrated with a series of donor (EGFP)-acceptor (mCherry) fusion proteins separated with linkers between 6-45 amino acids. CRX and NRL were fused at either terminus with EGFP or mCherry to create fluorescent proteins, and all combinations were tested in transiently transfected cells. FRET signals between CRX or NRL homo-pairs were highest with both fluorophores fused to the DNA binding domains (DBD), lower with both fused to the activation domains (AD), and not significant when fused on opposite termini. NRL had stronger FRET signals than CRX. A significant FRET signal between CRX and NRL hetero-pairs was detected when donor was fused to the CRX DNA binding domain and the acceptor fused to the NRL activation domain. FRET signals increased with CRX or NRL expression levels at a rate much higher than expected for collisional FRET alone. Together, our results show the formation of CRX-NRL complexes in live HEK293T cells that are close enough for FRET.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Factores de Transcripción
/
Transactivadores
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Proteínas de Homeodominio
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Transferencia Resonante de Energía de Fluorescencia
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico
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Proteínas del Ojo
Límite:
Humans
Idioma:
En
Revista:
Sci Rep
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos