News and Publications

Dynamics of Adhesion Signaling in Living Cells

Cytoplasm is an essentially continuous network of organized molecules, with large structures such as cytoskeletal "girders" and organelles forming a shifting scaffold for the organization of many smaller molecular assemblies. This structure is an important element in the regulatory circuitry of the cell; it controls the precise timing and location of molecular interactions that determine cell behavior. Such supramolecular organization is difficult to understand by examining isolated proteins in vitro. Therefore, we are developing and using new tools to visualize protein activities within individual, living cells.

Our techniques depend on novel dyes that "report" many aspects of protein behavior and on novel methods for site-specific attachment of these dyes to proteins and peptides. The dyes, which are optimized for use in vivo, show large changes in fluorescence that depend on their interaction with nearby amino acids or exposure to water. When attached to proteins, the dyes are affected by the binding of specific ligands, conformational changes, or phosphorylation.

We focused on building new biosensors that report the activation of MAP kinases and the Rho family of small GTPases. Each of these important signaling molecules is involved in multiple cellular behaviors (proliferation, apoptosis, motility), participating in essentially opposite processes through tight regulation of the timing and location of their activation. These proteins also generate an array of different cytoskeletal and morphologic changes, which are tightly controlled in time and space to produce motility and other polarized cell behaviors.

In one approach, we used protein domains and/or antibody fragments modified so that they generate a fluorescence signal when they find and bind to a protein with a particular conformation. In this way, we visualized localized GTP binding of each of the Rho GTPases (Rac, Rho, and Cdc42), showing that the enzymes are turned on with precise timing at different positions within a moving cell. These 3 GTPases participate in a signaling cascade to control one another's activation. By developing dyes of different wavelengths, we can systematically examine this entire signaling pathway in the same cell to see how the proteins regulate one another during rapid, precisely orchestrated behaviors.

We identified adhesion molecules, including vinculin, that affect the activation of the MAP kinases and Rho family molecules to shift the balance between apoptosis, motility, and proliferation. Speci-fic interactions between vinculin and its ligands affect the dynamics of MAP kinases and Rho proteins to generate different cell behaviors. We are now investigating (1) the mechanisms by which such dynamics control signals downstream of Cdc42 and Erk2 and (2) the cascades that coordinate the activity of the different Rho GTPases.

Publications

Gaits, F., Hahn, K. Shedding light on cell signaling: interpretation of FRET biosensors, Sci. STKE PE3, 2003, January 14 Issue.

Gardiner, E.M., Pestonjamasp, N., Bohl, B.P., Chamberlain, C., Hahn, K.M., Bokoch, G.M. Spatial and temporal analysis of Rac activation during live neutrophil chemotaxis: roles in leading edge extension and tail retraction. Curr. Biol. 12:2029, 2002.

Han, W.G., Liu, T., Himo, F., Toutchkine, A., Bashford, D., Hahn, K.M., Noodleman, L.M. Theoretical study of the UV absorption and emission solvatochromic properties of solvent-sensitive dyes. Chemphyschem, in press.

Toutchkine, A., Kraynov, V., Hahn, K. Solvent-sensitive dyes to report protein conformational changes in living cells. J. Am. Chem. Soc. 125:4132, 2003.

Tu, S.S., Wu, W.J., Yang, W., Nalbant, P., Hahn, K., Cerione, R.A. Antiapoptotic Cdc42 mutants are potent activators of cellular transformation. Biochemistry 41:12350, 2002.