New approaches will be developed to examine the dynamics of signaling protein activation in living cells. These methods will be based upon novel, environmentally-sensitive fluorescent dyes designed to respond to protein conformational changes in vivo. Generally applicable methods and dyes will be developed by designing biosensors for the nucleotide state of Cdc42, Rac, and Rho. The new approaches will provide the ability to study endogenous, unlabeled proteins in living cells, to study activation of multiple proteins in the same cell, and to examine proteins buried within multiprotein complexes. The dyes will be very bright and undergo large fluorescence changes, enabling us to use low biosensor concentrations for minimum perturbation of normal cell physiology. The dyes can be used to obtain many images before photobleaching, providing excellent temporal resolution, and quantitation of changes in protein activation level over time in individual cells. Biosensors of Rac and Cdc42 will be imaged in the same cell, to examine how Cdc42 activation of Rac coordinates the activity of the two GTPases for motility. Finally, we will develop means to convert antibody fragments into biosensors, providing access to many protein activities. The new dyes will make it possible to use highly reversible antibody binding, to minimally perturb cell behavior. A monoclonal antibody specific for phosphorylated alpha4 integrin will be used as a model for development of the approach, and to study alpha4 regulation of Rac in directed cell movement.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-SSS-U (10))
Program Officer
Deatherage, James F
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
Zip Code
Karginov, Andrei V; Hahn, Klaus M; Deiters, Alexander (2014) Optochemical activation of kinase function in live cells. Methods Mol Biol 1148:31-43
Tsygankov, Denis; Bilancia, Colleen G; Vitriol, Eric A et al. (2014) CellGeo: a computational platform for the analysis of shape changes in cells with complex geometries. J Cell Biol 204:443-60
Khalil, Bassem D; Hanna, Samer; Saykali, Bechara A et al. (2014) The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility. Exp Cell Res 321:109-22
Allen, R J; Tsygankov, D; Zawistowski, J S et al. (2014) Automated line scan analysis to quantify biosensor activity at the cell edge. Methods 66:162-7
Samson, Thomas; van Buul, Jaap D; Kroon, Jeffrey et al. (2013) The guanine-nucleotide exchange factor SGEF plays a crucial role in the formation of atherosclerosis. PLoS One 8:e55202
Hinde, Elizabeth; Digman, Michelle A; Hahn, Klaus M et al. (2013) Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM. Proc Natl Acad Sci U S A 110:135-40
Konze, Kyle D; Ma, Anqi; Li, Fengling et al. (2013) An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem Biol 8:1324-34
MacNevin, Christopher J; Gremyachinskiy, Dmitriy; Hsu, Chia-Wen et al. (2013) Environment-sensing merocyanine dyes for live cell imaging applications. Bioconjug Chem 24:215-23
Lungu, Oana I; Hallett, Ryan A; Choi, Eun Jung et al. (2012) Designing photoswitchable peptides using the AsLOV2 domain. Chem Biol 19:507-17
Hinde, Elizabeth; Digman, Michelle A; Welch, Christopher et al. (2012) Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy. Microsc Res Tech 75:271-81

Showing the most recent 10 out of 40 publications