Open AccessSensitive FRET Biosensor Reveals Fyn Kinase Regulation by Submembrane Localization
Author(s) -
Mingxing Ouyang,
Rongxue Wan,
Qin Qin,
Peng Qin,
Pengzhi Wang,
Jenny Wu,
Michael G. Allen,
Yiwen Shi,
Shan Laub,
Linhong Deng,
Shaoying Lu,
Yingxiao Wang
Publication year - 2018
Publication title -
acs sensors
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.055
H-Index - 57
ISSN - 2379-3694
DOI - 10.1021/acssensors.8b00896
Subject(s) - fyn , förster resonance energy transfer , microbiology and biotechnology , src family kinase , kinase , chemistry , biophysics , biology , proto oncogene tyrosine protein kinase src , fluorescence , physics , quantum mechanics
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.