News and Publications

Large-Scale Organization of Signaling: Fluorescent Sensors of Protein Behavior In Vivo

K.M. Hahn, S. Bark, C. Chamberlain, K. Hallermalm, V. Kraynov, R. Muthyala, S. Slabaugh, C. Subauste

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. These smaller molecular machines change composition and position during dynamic polarized cell behaviors or as placement of the machines within the cell is used to control interacting signaling pathways. Such supramolecular organization is difficult to understand by examining isolated proteins in vitro. We are developing new tools that will enable us to visualize and quantify protein structural changes and ligand interactions within living cells. In studies that we hope will shed light on the principles that govern large-scale signaling organization, the first tools are now being used to examine signaling by growth factors and control of cell morphology in apoptosis.

We are developing a series of indicators based on novel fluorescent dyes that can provide visual evidence of the polarity and charge of nearby molecules in vivo. When these dyes are placed on proteins, changes in the fluorescence of the dyes indicate (1) conformational changes caused by alterations in the position of nearby amino acids or (2) the presence of charged ligands or posttranslational modifications. We are refining these dyes and are also investigating new methods for site-specific protein labeling, because placement of the dyes is critical to the response of the protein analogs.

We are focusing on the Rho family of small GTPase signaling proteins. These proteins are ubiquitous switches that control a wide range of biological processes in the cell. Because the nucleotide-binding state of the proteins is the critical determinant of whether the proteins are "on" or "off," we have produced fluorescent protein analogs that indicate whether the Rho proteins are bound to GTP or to GDP in individual, living cells. With these indicators in hand, we are beginning to examine how the localization and kinetics of Rac and Cdc42 activation mediate cross talk between integrins and growth factors. In collaboration with G. Bokoch, Departments of Immunology and Cell Biology, we are also studying how these proteins produce finely orchestrated morphologic changes during apoptosis. In the course of this work, we discovered that Rho proteins not only control morphologic changes but also control the induction of apoptosis by cytotoxic T cells, key mediators of the immune response.

Finally, in collaboration with S. Schmid, Department of Cell Biology, we are developing sensors of the phosphorylation of receptors for growth factors. These sensors will be used to examine how signaling by growth factors is mediated by changes in the phosphorylation of receptors as the receptors traverse the endocytic pathway.

PUBLICATIONS

Ben-Ami, G., Ozeri, V., Hanski, E., Hofmann, F., Aktories, K., Hahn, K.M., Bokoch, G.M., Rosenshine, I. Agents that inhibit Rho, Rac, and Cdc42 do not block formation of actin pedestals in HeLa cells infected with enteropathogenic Escherichia coli. Infect. Immun. 66:1755, 1998.

Chuang, T.-H., Hahn, K.M., Lee, J.D., Danley, D.E., Bokoch, G.M. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes. Mol. Biol. Cell 8:1687, 1997.