Intracellular Signaling and Cytoskeletal Dynamics
In the Huse lab, we combine synthetic peptide chemistry, protein design, and fluorescence videomicroscopy in order to ask quantitative questions about intracellular signaling in lymphocytes.
We are particularly interested in:
- Intracellular signaling dynamics. We wish to understand the sensitivity and the kinetics of both activating and inhibitory signals, and how these signals interact.
- Cell biological responses to receptor activation, with particular emphasis on the polarization of the microtubule cytoskeleton.
- The development of methodology that enables quantitative, high-resolution analysis of signaling in single cells.
Signal transduction plays a central role in nearly every aspect of immune function, and has been the subject of intense biochemical and genetic analysis for a number of years. As a result, many of the cell surface receptors and intracellular proteins important for these processes have been identified and characterized. Despite this progress, however, our knowledge of how these molecules work together dynamically in the context of a complex cellular response is limited. In addition, how signaling kinetics and pathway sensitivity change over time is unclear.
We have adopted a reductionist approach to address these deficiencies. First, agonist ligands for specific cell surface receptors are engineered in order to provide control of a particular stimulus parameter. Then, controlled amounts of these ligands are used to stimulate lymphocytes in the context of an imaging experiment. Subsequent signaling responses are monitored using fluorescent probes for intracellular events like phosphorylation and calcium influx. For example, we have recently developed a photoactivatable peptide-major histocompatibility complex (pMHC) reagent that is non-stimulatory to T cells until it is irradiated with UV light. The ability to photoactivate this reagent beneath individual T cells during an imaging experiment has enabled us to analyze signaling kinetics and localization downstream of the T cell receptor (TCR) with unprecedented precision.
We are currently adapting this approach to analyze intracellular signaling in natural killer (NK) lymphocytes, which play a central role in immune responses to viruses and cancer by selectively killing infected or transformed cells. NK cell activity is governed by an array of diverse cell surface receptors, some which deliver activating stimulation, and others that inhibit responses. No single activating or inhibitory receptor appears to dominate over all others. Rather, studies suggest that NK cells evaluate all activating and inhibitory stimuli, and formulate effector responses based on the "balance" of these signals. How, though, is this balance struck? How much activating or inhibitory signal is required to trigger or block, respectively, a given downstream response? Furthermore, are there spatial and temporal parameters that constrain the interactions between signaling pathways? Using the methodology described above in combination with more standard approaches, we aim to develop a quantitative understanding of these issues, which should aid in the development of effective strategies for the control of NK cell activity in more complex systems.
We are also interested in cytoskeletal dynamics, in particular the polarization of the microtubule cytoskeleton in response to activating stimuli. The recognition of agonist ligands by a T cell or an NK cell on the surface of a target cell induces the formation of a tight intercellular junction called an immunological synapse. Within minutes, the microtubule organizing center (MTOC) of the lymphocyte, along with its associated Golgi apparatus, reorients to a position just beneath the contact site. MTOC polarization mediates the targeted delivery of both cytokines and cytolytic factors to the synapse, and therefore plays a crucial role in maintaining the specificity of both intercellular communication and target cell killing. The molecular events responsible for this process, however, remain mysterious. Using photactivatable pMHC, we can trigger MTOC reorientation in single T cells. The ability to control T cell polarity in this fashion will enable us to mechanistically dissect the events involved in MTOC movement. This will, in turn, contribute to our ability to harness and manipulate lymphocyte effector responses.