Our lab focuses on two synergistic areas: developing molecular tools to visualize and control signaling in living cells, and using these tools to ask how the spatio-temporal dynamics of protein activity govern signaling in vivo. Our biological studies center on clinically important cell behaviors - phagocytosis, platelet production, adhesion signaling and immune cell interactions.
Our imaging tools are focused on specific molecules for our biological studies, but we aim to produce broadly applicable approaches to visualize and control protein behavior. These include new biosensor designs that minimally perturb signaling, enabling us to visualize multiple proteins simultaneously, and means to control endogenous proteins with light. We are engineering allosteric networks in proteins to produce fully functional analogs that can be controlled with light or small molecules. By precisely generating localized signaling gradients and activation events, we ask quantitative questions about signaling dynamics in cell decision making.
We are grateful to be working closely with other laboratories who model signaling dynamics, develop novel microscopes, and quantify complex behavior in microscope images.
See the"get tools" page for information regarding available molecules and methods (biosensors, optogenetics, engineered small molecule responses, dyes and software).
Some recent projects:
Spatio-temporal Dynamics of GEF-GTPase Networks. Rho family GTPases are ubiquitous molecular switches that control extraordinarily diverse cellular processes. They are activated by guanine nucleotide exchange factors (GEFs) that are roughly 5-fold more numerous than the GTPases themselves, and integrate the multiple cellular inputs that together determine GTPase function. GEFs and GTPases form complex networks that are constituted transiently and locally for specific purposes. Biochemical, genetic, molecular, and structural analyses have unraveled a great deal about these pathways, but their spatio-temporal regulation can only be fully understood in the context of intact cells.
We are combining the visualization and control of GEF/GTPase networks with our collaborators' computational tools that extract network architecture from imaging data. Our new biosensors will report GEF activation by specific upstream inputs, activation of endogenous GEFs, and enable simultaneous imaging and photomanipulation of GEFs and GTPases. Specifically, we are addressing the role of GEF/GTPase networks in mechanotransduction, and exploring novel findings regarding the structure of signaling complexes at cell adhesions. This project includes development of new membrane-permeable dyes and small molecule reporters of protein function.
Geometrically Precise Signaling Networks in Living Cells. Modern microscopy and image analysis, together with fluorescent probe technology, have evolved to quantify signaling in living cells and animals with seconds and submicron resolution. More recently, optogenetics and chemogenetics have made it possible to control signaling in vivo, and thereby explore causal relationships among signaling molecules as they are regulated by spatio-temporal dynamics. We are looking at systems like phagocytosis and immunological synapses where signals occur with precise geometry and timing, and are therefore amenable to modeling and quantitative studies. Approaches include dye-based biosensors of endogenous protein conformation, engineered allosteric control for inhibition or activation by light, and the use of photoresponsive protein analogs that can serve as substitutes for endogenous proteins. We have developed means to study conformational changes of single molecules in living cells, and are using this to focus on adhesion dynamics and structure. Precise control of activation gradients and kinetics are being used to inform mathematical models examining how signaling domains are maintained and used.
Small GTPase Circuits in Platelet Formation. Megakaryocytes (MKs) are specialized blood cells that produce all of the platelets found in the human body. They reside primarily in the bone marrow, where they undergo proplatelet formation (PPF), a process characterized by dramatic changes to the cytoskeleton. Impaired platelet production leads to thrombocytopenia (low platelet count), which can cause life-threatening bleeding complications. Treating thrombocytopenia requires millions of platelet transfusions annually in the US alone. The high demand for platelet concentrates presents a significant problem in transfusion medicine, as platelets have a short shelf-life so must be supplied frequently by volunteer donors. Multifaceted approaches are under investigation to address this problem by producing platelets in vitro, but the low efficiency of PPF and the low yield of platelets remain major obstacles.
The overarching goal of our project is to elucidate the mechanism of PPF, and thereby develop strategies for carefully timed perturbations of Rap and/or Rho GTPase activity as a means to improve PPF efficiency and the quality of the platelet product. Rho-family GTPases are master regulators of the cytoskeleton that control morphodynamics through localized, precisely timed activation events. It has recently become clear that they are important regulators of PPF, but very little is known about their spatio-temporal dynamics or coordination. Our collaborators in the Bergmeier lab have shown that PPF requires signaling by the Ras-family GTPases Rap1A and Rap1B; mice deficient in both Rap1 isoforms in MKs show significant thrombocytopenia and a near complete loss of PPF in vitro. To elucidate how GTPases orchestrate the complex morphological changes of PPF, we are designing molecules to visualize and photo-manipulate GTPase activity, and developing microscope techniques to study biosensors and optogentic analogs in 3D. We are defining the contribution of Rap1 isoforms and their regulators to MK development and PPF, establishing GTPase “activity signatures” and network connections critical to PPF. We are pursuing precisely targeted perturbation of GTPase activity to optimize in vitro platelet production. We are trying to produce enabling technologies that can be applied by researchers in a wide range of fields.
Thanks to these organizations, charitable donors and the taxpayers for supporting our research over the years: National Institutes of Health, National Science Foundation, American Cancer Society, American Heart Association, The Leukemia and Lymphoma Society, Arthritis Foundation, Human Frontiers in Science Program, Department of Defense, NC Biotechnology Center, Deutsche Forschungsgemeinschaft, Autism Speaks, UNC's University Cancer Research Fund, UNC Lineberger Cancer Center, and the UNC Institute for Developmental Disabilities.
~ Updated 07/03/2019