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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21GM106403-03
Application #
8728967
Study Section
Special Emphasis Panel (ZRG1-CB-D (51))
Program Officer
Deatherage, James F
Project Start
2012-09-21
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$187,938
Indirect Cost
$66,688
Name
University of California San Diego
Department
Engineering (All Types)
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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
Kim, Tae-Jin; Sun, Jie; Lu, Shaoying et al. (2014) The regulation of ?-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs. Biomaterials 35:8348-56
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
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; Wang, Ning; Wang, Yingxiao (2013) Mechanotransduction at focal adhesions: from physiology to cancer development. J Cell Mol Med 17:597-604
Lin, Li-Jung; Grimme, Jill M; Sun, Jie et al. (2013) The antagonistic roles of PDGF and integrin ?v?3 in regulating ROS production at focal adhesions. Biomaterials 34:3807-15