News and Publications

The Spatiotemporal Dynamics of Signaling in Living Cells

K. Hahn, F. Gaits, S. Grahn, S. Junger, P. Nalbant, O. Pertz, C. Subauste, A. Toutchkine

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. We are developing new tools and using them 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 posttranslational modifications.

We focused on building new biosensors that report the activation of MAP kinases and the Rho family of small GTPases. These important signaling molecules are essential for very different cellular behaviors (proliferation, apoptosis, motility) and participate 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 activation of the MAP kinases and Rho family molecules to shift the balance between apoptosis and motility or proliferation. Specific interactions between vinculin and its ligands affect the dynamics of MAP kinases and Rho proteins to generate different cellular behaviors. The mechanisms by which such dynamics control signals downstream of Cdc42 and Erk2 are now being investigated.

PUBLICATIONS

Del Pozo, M.A., Kiosses, W.B., Alderson, N.D., Meller, N., Hahn, K.M., Schwartz, M.A. Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat. Cell Biol. 4:232, 2002.

Frosst, P., Guan, T., Subauste, C., Hahn, K., Gerace, L. Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export. J. Cell Biol. 156:617, 2002.

Hahn, K., Toutchkine, A. Live-cell fluorescent biosensors for activated signaling proteins. Curr. Opin. Cell Biol. 14:167, 2002.

Kiosses, W.B., Hahn, K.M., Giannelli, G., Quaranta, V. Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7. Cell Commun. Adhes. 8:29, 2001.

Nishimura, N., Pluttner, H., Hahn, K.M., Balch, W.E. The d subunit of AP-3 is required for efficient transport of VSV-G from the trans-Golgi network to the cell surface. Proc. Natl. Acad. Sci. U. S. A. 99:6755, 2002.

Subauste, M.C., List, B., Guan, X., Hahn, K.M., Lerner, R., Gilula, N.B. A catalytic antibody produces fluorescent tracers of gap junction communication in living cells. J. Biol. Chem. 276:49164, 2001.

Toutchkine, A., Nalbant, P., Hahn, K.M. Facile synthesis of thiol-reactive Cy3 and Cy5 derivatives with enhanced water solubility. Bioconjug. Chem. 13:387, 2002.

Weiss, T.S., Chamberlain, C.E., Takeda, T., Lin, P., Hahn, K.H., Farquhar, M.G. Gai3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins. Proc. Natl. Acad. Sci. U. S. A. 98:14961, 2001.