The discovery and development of genetically-encoded fluorescent proteins (FPs) have revolutionized biology and medicine by allowing the visualization of molecular localization in live cells and animals. A general and high-efficiency strategy for developing and optimizing FRET-based biosensor can transform the field of biosensor development and allow the detailed investigation of different intracellular molecules, for the purpose of understanding and treating human diseases. Fyn kinase plays crucial roles in regulating a variety of important biological functions, including T cell receptor signaling, learnig and memory, as well as cell adhesion, mechanotransduction, and prostate cancer development. Therefore, we propose to develop a systematic strategy based on directed evolution to develop and optimize a Fyn FRET biosensor for the visualization of the subcellular Fyn activity in live cells, utilizing a high-efficiency FRET pair. Accordingly, two specific aims are proposed: (1) Optimize a substrate peptide sequence for Fyn kinase;(2) Develop an optimized SH2 domain and Fyn FRET biosensor. This systematic and directed evolution method can be readily extended for the development of, in principle, any FP-based genetically-encoded FRET biosensor. The information obtained should also advance our in-depth understanding of the molecular mechanism underlying the Fyn regulation. Hence, by integrating cutting-edge technologies in molecular engineering and live cell imaging, the success of the proposal should serve as a starting point to revolutionize the development of FRET biosensors and have transformative impact on biological studies in general.

Public Health Relevance

Fyn kinase plays crucial roles in regulating a variety of important pathophysiological processes, including T cell receptor signaling, learning and memory, as well as cell adhesion, mechanotransduction, and prostate cancer development. This proposal will integrate the cutting-edge directed evolution and FRET to systematically develop biosensors to visualize Fyn activity in live cells to advance our systematic understanding of the molecular mechanism underlying the Fyn regulation. Therefore, the success of the proposed project will have significant impact on improving public health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory/Developmental Grants (R21)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-CB-D (51))
Program Officer
Deatherage, James F
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Diego
Engineering (All Types)
Schools of Arts and Sciences
La Jolla
United States
Zip Code
Nishitani, Wagner Shin; Alencar, Adriano Mesquita; Wang, Yingxiao (2015) Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion. PLoS One 10:e0126440
Kim, Tae-Jin; Joo, Chirlmin; Seong, Jihye et al. (2015) Distinct mechanisms regulating mechanical force-induced Ca²⁺ signals at the plasma membrane and the ER in human MSCs. Elife 4:e04876
Kim, Tae-Jin; Sun, Jie; Lu, Shaoying et al. (2014) The regulation of β-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs. Biomaterials 35:8348-56
Kim, Tae-Jin; Sun, Jie; Lu, Shaoying et al. (2014) Prolonged mechanical stretch initiates intracellular calcium oscillations in human mesenchymal stem cells. PLoS One 9:e109378
Lei, Lei; Lu, Shaoying; Wang, Yi et al. (2014) The role of mechanical tension on lipid raft dependent PDGF-induced TRPC6 activation. Biomaterials 35:2868-77
Seong, Jihye; Wang, Ning; Wang, Yingxiao (2013) Mechanotransduction at focal adhesions: from physiology to cancer development. J Cell Mol Med 17:597-604
Qian, Tongcheng; Lu, Shaoying; Ma, Hongwei et al. (2013) FRET imaging of calcium signaling in live cells in the microenvironment. Integr Biol (Camb) 5:431-8
Seong, Jihye; Tajik, Arash; Sun, Jie et al. (2013) Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins. Proc Natl Acad Sci U S A 110:19372-7
Sun, Jie; Lu, Shaoying; Ouyang, Mingxing et al. (2013) Antagonism between binding site affinity and conformational dynamics tunes alternative cis-interactions within Shp2. Nat Commun 4:2037
Lin, Li-Jung; Grimme, Jill M; Sun, Jie et al. (2013) The antagonistic roles of PDGF and integrin αvβ3 in regulating ROS production at focal adhesions. Biomaterials 34:3807-15

Showing the most recent 10 out of 12 publications