A fundamental property of living cells is their ability to respond and adapt to stimuli, yet we have only begun to appreciate cell decision-making processes at the molecular level. These mechanisms are known as signal transduction networks. While a general understanding of how intracellular signaling molecules interact in pathways has evolved in recent years, we are still unable to predict and control cell responses quantitatively under various conditions. The central tenet of our research is that the ability to manipulate cell behavior uniquely follows from understanding signal transduction as a complex chemical system.Our interdisciplinary approach, which combines mathematical modeling and analysis with molecular biology, cell biochemistry, and fluorescence imaging methods, has implications for cancer, immune regulation, and wound healing.
Molecular Crosstalk in Life and Death Signaling
Signaling pathways seldom operate in isolation. Interactions between molecules in different pathways imply the existence of a distributed signaling network that serves as a system of checks and balances; however, multiple mutations can combine to short-circuit the system, forming the molecular basis for cancer and other diseases. Further, interventions that target intracellular enzymes can have effects that propagate through the network, affecting drug efficacy. In particular, specific signaling pathways are required for cell proliferation and survival of many cell types, and the extensive crosstalk between these pathways suggests that cell life and death are co-regulated.
We are currently studying such networks in fibroblasts stimulated with platelet-derived growth factor (PDGF) and in T cells stimulated with interleukin-2 and -4; these model systems are important for tissue homeostasis and the immune response, respectively. We have developed quantitative, high-throughput biochemical assays to measure activation of the key molecular intermediates, and both genetic and pharmacological approaches are used to manipulate their activation states independently from the extracellular stimulus. This allows us to `open’ the control structure of the network and isolate specific intermolecular interactions. Kinetic models unify our observations and predict the effects of molecular interventions in combination, and pathway outcomes are correlated with cell proliferation and survival metrics to elucidate powerful design principles for engineering the cell life and death switch at the molecular level.
Intracellular Gradients and Directed Cell Migration
In wound healing, PDGF is secreted by platelets as they clot blood vessels. This stimulates directed migration of fibroblasts from connective tissue to the wound, where they secrete, remodel, and contract the extracellular matrix, rebuilding the tissue. Animal cells detect chemical gradients by spatial sensing, in which a cell can differentiate signaling at its front from its rear. We have demonstrated that PDGF gradients stimulate asymmetric production of specific lipid second messengers in the cell membrane, which apparently act as a cellular compass to signal migration in the appropriate direction.
We use total internal reflection fluorescence microscopy (TIRFM) to quantitatively image the production, lateral diffusion, and turnover of these membrane lipids in individual, living cells in real time and at ~100 nm resolution. This technique is used in conjunction with reaction-diffusion models that allow us to parse out these concurrent molecular processes under uniform and gradient stimulation with PDGF. We are currently extending this approach to stimulation with both soluble and surface-immobilized factors, to examine the relationships among intracellular signaling, cell-substratum adhesion, and the speed and orientation of cell migration. A quantitative understanding of spatial sensing will pave the way for novel wound healing therapies, with controlled delivery of PDGF and signal transduction-modifying agents to optimize the migration and proliferation of effector cells.
Focus Areas - Biomedical and Biochemical Engineering. Signal Transduction Networks. Mammalian Cell Engineering.