News and Publications

Live-Cell Biosensors and the Organization of Signaling in Living Cells

K. Hahn, C. Chamberlain, S. Grahn, S. Junger, V. Kraynov, P. Nalbant, C. Subauste, A. Toutchkine

Each cell contains 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 scaffold is an important element in the regulatory circuitry of the cell, controlling 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. We are developing and using new tools to visualize protein activities within individual, living cells.

The new techniques we are developing depend on novel dyes that can "report" many aspects of protein behavior and on novel methods for site-specific attachment of these dyes to proteins and peptides. The dyes are specifically designed for use in living cells; they show large changes in fluorescence properties that depend on the hydrophobicity of surrounding molecules or on specific interactions such as hydrogen bonding. We used dyes attached to proteins, including Erk and the small GTPase Cdc42, to investigate the binding of specific ligands and/or conformational changes. In collaboration with D. Burton, Department of Immunology, and P. Dawson, Department of Cell Biology, we are exploring new chemistries for site-specific labeling and rapid "combinatorial" screening of dye positions that can be used to report protein behavior without perturbing normal protein function.

Using protein domains and/or antibody fragments as biosensors that can report the binding of specific targets in living cells, we quantified the localized activation of small GTPase proteins. The Rho family of GTPases lies at the crossroads of many signaling pathways. Each member of the Rho family can produce several, essentially opposite cell behaviors. For example Rac and Cdc42 can induce either programmed cell death (apoptosis) or motility and proliferation. A cell can use the same protein to induce many different behaviors by precisely regulating the location and level of the protein's activation. By determining the localization and kinetics of activation of members of the Rho family, we hope to shed light on how this previously inaccessible level of control is used.

In addition to examining how cellular activities are regulated through the localization and kinetics of protein activity, we are focusing on the localized signals required to produce highly polarized cell behaviors. When a cell moves, different intricate machinery is built up at multiple specific sites. The front of the cell is specialized for extension, complex adhesion plaques attach the cell to the substrate and constantly change as the cell moves forward, and the rear of the cell contains contractile fibers specialized for detaching the cell as it moves. How are localized signals used to build up these machines, and how are signals coordinated to operate in synchrony? We are focusing on the role of the GTPases in generating many different types of actin cytoskeletal behavior and on the long-range communication mediated by these enzymes through lipid signaling pathways.

Finally, we discovered a new role in signaling for the protein vinculin, a component of adhesion complexes previously thought to play a primarily structural role. We found that vinculin regulates Erk, a protein that controls proliferation, movement, and apoptosis. Vinculin, Erk, and the Rho GTPases are all involved in polarized movement. We hope that by studying these molecules, we can uncover new mechanisms for long-range communication between cell components.

PUBLICATIONS
Bark, S.J., Schmid, S., Hahn, K.M. A highly efficient method for site-specific modification of peptides after chemical synthesis. J. Am. Chem. Soc. 122:3567, 2000.

Chamberlain, C.E., Hahn, K.M. Watching proteins in the wild: Fluorescence methods to study protein dynamics in living cells. Traffic 1:755, 2000.

Chamberlain, C.E., Kraynov, V., Hahn, K.M. Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. Methods Enzymol. 325:389, 2000.

Kiosses, W.B., Hahn, K.M., Giannelli, G., Quaranta, V. Characterization of laminin-5 morphological and cytoskeletal changes in MCF10A breast epithelial cells. Cell Adhes. Commun., in press.

Kraynov, V.S., Chamberlain, C.E., Bokoch, G.M., Schwartz, M.A., Slabaugh, S., Hahn, K.M. Localized Rac activation dynamics visualized in living cells. Science 290:333, 2000.

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