research

The cell is a continuous network of interacting molecules, constantly changing on many scales of size and time. In response to a subtle balance of extracellular cues, cellular networks undergo precisely orchestrated dynamics to produce cell behaviors. To understand how network dynamics integrate extracellular signals, we are developing molecular tools to view and manipulate protein behavior in living cells and animals. We use protein engineering and organic synthesis to develop molecules whose fluorescence reflects protein structural changes, and molecules that can be controlled within cells using light or engineered allosteric responses. These are coupled with our collaborators’ development of new modes of microscopy and novel computational approaches to model the spatio-temporal dynamics of signaling networks. This collaborative and synergistic approach enables us to probe dynamic network behaviors that are essential to cell function and can only be fully understood by studying molecular activity in living systems.

Transendothelial migration. During inflammation, leukocytes interact with the endothelium through ligation of adhesion molecules, enabling neutrophils to migrate over the endothelial surface as a prelude to transendothelial migration (TEM).  Molecules of the endothelium serve not only as adhesive ligands, but their engagement generates signals that promote neutrophil TEM through activation of multiple Rho GTPases. Little is known about how these are activated or how they act sequentially and synergistically to coordinate the cytoskeletal dynamics needed for neutrophils to exit blood vessels. Our recently developed techniques for examining multiple biosensors simultaneously are enabling us to see how these proteins work coordinately for TEM. Novel photomanipulation techniques are being used to activate or inhibit specific GTPases and other molecules at precise times and places in the endothelial cells. Using 3D force microscopy developed by our collaborator, Richard Superfine, we are asking how physiologically relevant forces affect adhesion molecule signaling to Rho family GTPases. Understanding how these processes are co-ordinated and regulated is critical for identifying new therapeutic targets that maximize benefits of host defense and minimize endothelial damage, particularly in the lungs where edema interferes with gas exchange. (Image: Leucocytes induce cups in endothelial cells for transendothelial migration; Jaap Schroeder, Burridge Lab, UNC)

Hahn lab members: Janet Dolittle, Ellen O’Shaughnessy, Scott Slattery, Hui Wang
Collaborators: Burridge, Doerschuk, and Superfine labs, all at UNC-Chapel Hill

Visualization and photomanipulation of signaling in vivo.We are developing new tools to quantify and manipulate signaling in living animals, with an initial focus on polarized motility and zebrafish models of cancer.  The use of fluorescent biosensors, and more recently photomanipulation of protein activity, has generated a revolution in cell biology. However, it has been challenging to apply these tools in live animals to study tissue dynamics and the trafficking of immune cells. Our recent studies highlighted the development and application of novel optogenetic techniques to manipulate and analyze cell movements in vivo during zebrafish development, Drosophila development and cocaine addiction in mice. We have developed new biosensor designs that can report conformational changes and phosphorylation of endogenous proteins. By building on this work, we are generating new approaches that can be applied broadly for insight into tissue and organ physiology in live animals. We are using these tools to address epithelial to mesenchymal transition (EMT) and immune surveillance in cancer.  The long term goal of this work is to bring the revolution in cell biology and cell signaling in vivo, thereby enabling application to broad areas of developmental biology and disease pathogenesis.
(Image: Activation of RhoA activity by localized irradiation of photoactivatable Rac; Yi Wu, Hahn Lab)

Hahn lab members: Onur Dagliyan, Andrei Karginov, Marie Rougie, Hui Wang, Jason Yi
Collaborators:  Huttenlocher lab, U. Wisconsin and Kevin Elicieri at LOCI, U. Wisconsin

Dynamics of GEF-GTPase networks. This is a large project involving interactions between multiple laboratories.To study the networks of GEF and GTPase proteins that control a broad array of cell functions, we have combined forces with the Sondek, Danuser, Hall and Burridge laboratories to are develop new biosensors, protein photomanipulation tools and  computational methods for analyzing network dynamics in vivo. These  are being applied  to study mechano-transduction, sheet migration and tissue morphodynamics. In our own biological studies we are visualizing and quantifying the precisely localized signals that control cell polarization, including dynamic feedback and feedforward loops that can only be fully understood in vivo. By imaging the conformational changes of endogenous, untagged proteins we can examine subtle constitutive fluctuations in signaling pathways. These small fluctuations reflect real physiological interactions rather than the large perturbations  introduced by hyperactivation of individual receptors.  This work will ultimately be extended to examine upstream regulators and downstream effectors of the GTPases, extending from receptor stimulation to the cytoskeleton and adhesion complexes. (Image: Biosensor fluctuations reveal GEF/GTPase feedback loops in cell migration; Hunter Elliott, Danuser Lab, Harvard)

Hahn lab members: Li Li, Dan Marston, Brian Mehl, Scott Slattery, Hui Wang
Collaborators: Danuser lab at Harvard, Hall lab at Sloan Kettering Inst., Burridge and Sondek labs at UNC-Chapel Hill.

Engineered allosteric regulation of protein activity.
By understanding the structural and dynamic underpinnings of allosteric regulation we are learning to engineer allosteric regulation into proteins, rendering them susceptible to activation or inhibition by small molecules. Controlling cell behavior through engineered allosteric regulation has great potential for therapeutic intervention in humans, for fundamental research, and for controlling proteins in animal models. We recently demonstrated a new technique to regulate the catalytic activity of kinases through insertion of an engineered allosteric switch. The altered kinases were inactive until cells were treated with a membrane permeable small molecule. Based on this work we are now developing orthogonal regulation of multiple targets, and activation of specific signaling interactions. Our collaborator Dr. Dokholyan is developing computational methods  to identify allosteric interaction networks in proteins; through this analysis we are exending the approach to other protein families.  Engineered allosteric activation is being used to dissect the role of specific protein isoforms in cell polarization and cancer.  (Image: Allosteric switch inserted in focal adhesion kinase; courtesy Dokholyan Lab, UNC)

Hahn lab members: Pei-Hsuan Chu, Onur Dagliyan, Andrei Karginov
Collaborators: Der, Dokholyan and Elston labs, all at UNC-Chapel Hill

Thanks to our collaborators for sharing their enthusiasm and for making exciting science possible. Here are the web sites of some of our current collaborators:

• Backer Laboratory, Albert Einstein College of Medicine
• Barber Lab, UCSF
• Bourne Laboratory, UCSF
• Burridge Lab, UNC
• Condeelis Lab, Albert Einstein College of Medicine
• Danuser Lab, Harvard
• Devreotes Lab, Johns Hopkins University
• Dokholyan Lab, UNC
• Doerschuck Lab, UNC
• Elston Lab, UNC
• Gomez Lab, UNC
• Gratton Lab, NIH Laboratory for Fluorescence Dynamics, UC-Irvine
• Hodgson Lab, Albert Einstein College of Medicine
• Huttenlocher Lab, U. Wisconsin
• Jacobson Lab, UNC
• Johnson Lab, UNC
• Kasai Lab, U. Tokyo
• Kay Lab, U. Illinois-Chicago
• Kuhlman Lab, UNC
• LOCI at U. Wisconsin, Kevin Elicieri, Director
• Loew Lab, Center for Cell Analysis and Modeling, UConn
• Montell Lab, Johns Hopkins University
• Nalbant Lab, Germany
• Panomics
• Protein Structure Initiative, Albert Einstein College of Medicine
• Schwartz Lab, Yale
• Sondek Lab, UNC
• Superfine Lab, NIH Center for Computer Integrated Systems for Microscopy and Manipulation, UNC

Thanks to the following organizations, and to the US and NC taxpayers, for their support: