The Integration of FLIM and FRET for the Visualization of Flow-induced Molecular Hierarchies The research activity of the PI's laboratory is focused on the integration of cutting edge technologies for the elucidation of molecular mechanism in vascular biology, with the ultimate goal of enhancing our knowledge on cardiovascular functions and contributing to the health and well-being of humankind. In particular, novel imaging biosensors and technologies will be developed and employed to visualize the signaling transduction with high spatiotemporal resolutions in live cells under different mechanical forces, e.g. shear stress. In the long run, we aim to apply the knowledge and insights gained via these novel technologies for the development of new strategies and reagents to treat cardiovascular diseases, including thrombosis and atherosclerosis. Along this line, we proposed to apply biosensors based on fluorescence resonance energy transfer (FRET) to visualize the Src activity and its related molecular hierarchy at sub-cellular levels during mechanotransduction, which is funded by NHLBI (R01HL098472). Since FAK activity and its autophosphorylation at tyrosine 397 are critical for the Src activation, we plan to investigate the spatiotemporal coupling between FAK and Src activations in endothelial cells under mechanical stimulation. Recently, we have developed a new FRET pair, mOrange2 and mCherry, which contains colors distinct from the popular FRET pair, CFP and YFP. As such, two different molecular events, such as the activities of FAK and Src, can be simultaneously monitored in the same live cell with biosensors based on mOrange2/mCherry and CFP/YFP. However, there is a certain overlap between the excitation spectra of mOrange2 and mCherry to cause a non-specific cross- excitation of mCherry, which can cause artifacts and lower the sensitivity of FRET biosensors. Fluorescence lifetime imaging microscopy (FLIM) could help to solve this problem because the emission lifetime measurement of the donor mOrange2 alone is sufficient to deduce the FRET efficiency, without the need to measure the emission lifetime of the acceptor mCherry. The main objective of the current proposal is hence for the PI to obtain in-depth training in optics and fluorescence, particularly FLIM and its associated instruments and imaging analysis. Part of the training requires the PI to attend formal courses and training programs whereas the other aspects of training involve more self-studying and interactions with collaborators. Accordingly, three specific aims are proposed for the research plan: (1) develop a mOrange2/mCherry-based FAK biosensor and characterize it by FLIM;(2) visualize the spatiotemporal map of FAK activity under different flows with FLIM;(3) simultaneously monitor FAK and Src activities under different flows with FLIM. The information obtained will significantly advance our systematic understanding of the molecular mechanism by which different flows affect cardiovascular diseases, such as atherosclerosis and restenosis. The newly developed biosensors will also provide powerful tools for detecting cardiovascular diseases and assessing the efficacy of therapeutic inhibitors.

Public Health Relevance

Endothelial cells (ECs) are continuously exposed to flow and its resultant shear stress, which plays crucial roles in regulating EC function and the ensuing patho-physiological processes, such as atherosclerosis and restenosis. This proposal will allow the PI to obtain in-depth training in optics and fluorescence, particularly FLIM and its associated instruments and imaging analysis, such that the PI can integrate FRET and FLIM to visualize FAK and Src activities simultaneously in live ECs to advance our systematic understanding of the molecular mechanism of mechanotransduction. Therefore, the success of the proposed project will have significant impact on improving public health.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Scientist Development Award - Research (K02)
Project #
7K02HL109142-03
Application #
8460536
Study Section
Special Emphasis Panel (ZHL1-CSR-K (M4))
Program Officer
Carlson, Drew E
Project Start
2011-08-15
Project End
2016-04-30
Budget Start
2013-05-24
Budget End
2014-04-30
Support Year
3
Fiscal Year
2013
Total Cost
$109,828
Indirect Cost
$8,135
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
Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat et al. (2016) Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound. Sci Rep 6:20477
Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye et al. (2016) In-situ coupling between kinase activities and protein dynamics within single focal adhesions. Sci Rep 6:29377
Li, Kaitao; Xiang, Xue; Sun, Jie et al. (2016) Imaging Spatiotemporal Activities of ZAP-70 in Live T Cells Using a FRET-Based Biosensor. Ann Biomed Eng 44:3510-3521
Gomez-Godinez, Veronica; Preece, Daryl; Shi, Linda et al. (2015) Laser-induced shockwave paired with FRET: a method to study cell signaling. Microsc Res Tech 78:195-9
Chung, Eddie Y; Ochs, Christopher J; Wang, Yi et al. (2015) Activatable and Cell-Penetrable Multiplex FRET Nanosensor for Profiling MT1-MMP Activity in Single Cancer Cells. Nano Lett 15:5025-32
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
Zhuo, Yue; Qian, Tongcheng; Wu, Yiqian et al. (2015) Subcellular and Dynamic Coordination between Src Activity and Cell Protrusion in Microenvironment. Sci Rep 5:12963
Kim, Tae-Jin; Zheng, Shuai; Sun, Jie et al. (2015) Dynamic visualization of α-catenin reveals rapid, reversible conformation switching between tension states. Curr Biol 25:218-24
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

Showing the most recent 10 out of 28 publications