Atherosclerosis, a disease represented by the lumen narrowing of arteries due to plaque formation, is one of the leading causes of death in the United States and most other developed countries. Atherosclerosis occurs likely at vascular branch sites where the vessel walls are exposed to disturbed flow, but not at the straight parts of vessels where laminar flow dominates. Evidence has shown that different flows and the ensuing shear stresses play crucial roles in regulating vascular endothelial cells (ECs), and subsequently endothelium permeability and atherosclerosis. However, it is unclear about the detailed shear stress distribution on EC surface and how ECs sense the spatiotemporal features of these mechanical stimuli to determine patho-physiological outcomes. In this proposal, the PI will develop an elastic molecular biosensor to visualize the shear stress distribution on live EC surfaces, utilizing the fluorescence resonance energy transfer (FRET). A new intracellular molecular biosensor will also be developed. Hence, both the signals of extracellular shear stress and intracellular molecular activity can be visualized in a single live cell. Three specific aims for this career proposal have been proposed: (1) To develop and characterize a FRET-based shear stress biosensor in vitro; (2) To visualize the distribution of shear stress on live ECs under different flow patterns; (3) To visualize extracellular shear stress and intracellular molecular activities simultaneously under different flow patterns. Given the importance of shear stress in endothelium permeability, and subsequently atherosclerosis, the significance of the proposed study can provide new information on the molecular mechanism by which cells sense the spatiotemporal properties of mechanical stimuli and convert them into biochemical/physiological signals. The main educational objective of this proposal is to motivate the high school and undergraduate students toward future career goals as the next generation of leading scientists in bioengineering. The PI will combine the proposed research and education and organize workshops for undergraduate students, featuring live cell imaging, bio-nanotechnology, and mechanobiology. Minorities and women will be particularly encouraged to participate in these workshops so that they are exposed to the fundamental principles and exciting advancement in these interdisciplinary fields. A significant effort will also be invested to bring the fascinating research advancement in the laboratory to the younger generation, i.e. high-school students. Research experiments will be videotaped and distributed through the internet to reach a wide audience of high-school teachers and students. An emphasis will be placed on how the mechanical and biochemical properties of cell can be monitored, measured, manipulated, and modeled to help our understanding of the complex biological system. The ultimate goal of the proposed research is to help the development of new tools for the early diagnosis of diseases related to mechanical force and the improvement of the treatment. The educational impact is to motivate the next generation of students, particularly underrepresented groups, to participate and promote the emerging field of bioengineering, and to bring broad impact to the society within and beyond the university.
1. We have constructed a new biosensor based on fluorescence resonance energy transfer (FRET). We have been engineering a shear stress biosensor based on fluorescence resonance energy transfer (FRET). The functional domain of this biosensor consists of a vWF A1 domain, an ECFP as FRET donor, a long linker, a YPet as FRET acceptor, and a GPIBa. We have engineered different biosensors varying the linker, the binding pair GPIBa and vWF A1 domain. When the biosensors are purified and coated on the beads, we can observe and compare the FRET signals. The results indicate that a shorter linker can cause a higher FRET signal, as predicated. These biosensors will be applied to detect the surface shear stress distribution upon flow application. 2. We visualized a polarized distribution of Src/FAK activation in endothelial cells upon shear stress application, which is dependent on RhoA and membrane fluidity. A polarized Src activation was observed with higher activity at the side facing the flow, which was enhanced by a cytochalasin D-mediated disruption of actin filaments but inhibited by a benzol alcohol-mediated enhancement of membrane fluidity. These results indicate that the flow-induced Src and subsequently FAK polarity depends on the coordination between intracellular tension distribution regulated by RhoA, its related actin structures and the plasma membrane fluidity. (Liu, et al, Nature Scientific Reports, 2014, Accepted). 3. We developed a software package capable of quantifying the spatiotemporal correlation between multiple molecular signals (Src activity and paxillin disassembly) in a single live cell based on FRET imaging. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics (Lu S et al, Nature Scientific Reports, 2014). In conclusion, we have optimized and characterized a new FRET biosensor capable of detecting shear stress. Substantial progress in our project was achieved by elucidating the molecular mechanism by which endothelial cells regulate the shear-induced Src and FAK activation in space and time. 4. Outreach Educational Activities: My lab members and myself continued to participate for various community activities, workshops, and symposiums aiming to educate the next generation of young scientists. These activities include (1) the 5th Sino-American Workshop on Biomedical Engineering and China-Oversea Joint Workshop on Biomechanics in Beijing China, Aug 2013; (2) SoCal SysBio Conference at University of California, Irvine, in Jan 2014; (3) International Workshop on Multiscale Mechanobiology at Hong Kong, in May 2014; (4) IEEE Nanobiosensing Workshop at Chicago in Aug 2014; (5) At Carmel Valley Middle School Math League (2014), we have lectured to 40 kids at 7-8 grade level. 5. In the past year, we have 7 Peer-Reviewed Journal papers published in related research: Liu B., et al, Wang Y. (2014) (Nature) Scientific Reports, Accepted Hu Y.-L., et al, Wang Y., Lasheras J.C., Chien S. (2014) (Nature) Scientific Reports, Accepted Lu S., et al, and Wang Y. (2014) (Nature) Scientific Reports, Accepted Kim T.J., et al, Wang Y (2014) PLoS ONE, Accepted Kim TJ, et al, Wang Y (2014) Biomaterials, Accepted Hum JM., et al, Wang Y. Pavalko FM (2014) PLoS ONE, Accepted Seong J., et al, Wang Y. (2013) PNAS, 110(48):19372-7 6. Broad Impact: The biosensors and constructs developed in this project have been distributed to other researchers upon request across the world. These biosensors allowed Dr. Chang Lu’s lab at Virginia Tech to monitor Src activities in single cells (1); Dr. Shyni Varghese at UCSD for the visualization of cancer cell invasion into extracellular matrix protein gels (2); Dr. Ning Wang’s lab at UIUC to discover a dynamic histone methylation in cancer cell development (3); Dr. Pavalko at Indiana University to detect the mechanotransduction processes in live bone cells (4); Dr. Paul Timpson at Garvan Institute of Medical Research in Sydney Australia for the screening of inhibitors on cancer invasion (5). Sun C., et al, Wang Y. and Lu C. (2014) Chem Comm, Accepted Aung A., et al, Wang Y., Jamora C., del Alamo JC, and Varghese S. (2014) Biophys. J., Accepted Tan Y., et al., Wang Y., Huang B., Wang N. (2014) Nature Communications, Accepted Hum JM., et al, Wang Y. Pavalko FM (2014) PLoS ONE, Accepted Nobis M, et al, Wang Y, et al, Anderson KI. (2013) Cancer Research, 73(15):4674-86
