Sensitive FRET Biosensor Reveals Fyn Kinase Regulation by Submembrane Localization.
ACS Sens
; 4(1): 76-86, 2019 01 25.
Article
em En
| MEDLINE
| ID: mdl-30588803
ABSTRACT
Fyn kinase plays crucial roles in hematology and T cell signaling; however, there are currently limited tools to visualize the dynamic Fyn activity in live cells. Here we developed and characterized a highly sensitive Fyn biosensor based on fluorescence resonance energy transfer (FRET) to monitor Fyn kinase activity in live cells. Our results show that Fyn kinase activity can be induced in both mouse embryonic fibroblasts (MEFs) and T cells by ligand engagement. Two different motifs were further introduced to target the biosensor at the cellular membrane microdomains in MEFs, revealing that the Fyn-tagged biosensor had 70% greater response to growth factor stimulation than the Lyn-tagged version. This suggests that the plasma membrane microdomains can be categorized into different functional subdomains. Further experiments show that while the membrane accessibility is necessary for Fyn activation, the localization of Fyn outside of its microdomains causes its hyperactivity, indicating that membrane microdomains provide a suppressive microenvironment for Fyn regulation in MEFs. Interestingly, a relatively high Fyn activity can be observed at perinuclear regions, further supporting the notion that the membrane microenvironment has a significant impact on the local molecular functions. Our work hence highlights a novel Fyn FRET biosensor for live cell imaging and its application in revealing an intricate submembrane regulation of Fyn in live MEFs.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
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Microdomínios da Membrana
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Transferência Ressonante de Energia de Fluorescência
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Proteínas Proto-Oncogênicas c-fyn
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article