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Doug LauffenburgerBiosketchBS Chemical Engineering, University of Illinois, 1975 Research summaryResearch in the Lauffenburger laboratory aims generally at a quantitative and integrative understanding of the molecular regulatory networks – primarily, although not exclusively, proteomic signaling — that regulate cell phenotypic behaviors (e.g., death, proliferation, migration, differentiation, and protein secretion) in response to environmental stimuli. We are especially interested in constructing and validating computational models that predict how molecular interventions may beneficially modulate cell behavior. Efforts under auspices of the CDP Center program continue to focus on mammalian epithelial cell responses (predominantly hepatocytes) within inflammatory contexts, and development of advances in experimental and computational methods for understanding these responses. Three main project avenues are currently being pursued. In project avenue one, over the past year we have been collaborating with the Han laboratory to create microdevice technology capable of measuring kinase activities on a single-cell basis. Although other methods exist for measuring phosphoprotein levels on a single-cell basis (e.g., flow cytometry, fluorescence microscopy), we have learned from past CDP Center work that kinase activity measurements generally contain greater information content for predicting cell phenotypic responses. Moreover, we seek to measure these vital signaling properties in direct association with the cellular phenotypic behavior. We have constructed a combination of a single-cell lysis probe and a nanofluidic protein concentrator, and recently demonstrated measurement of MK2 activity on a fluorescent substrate reporter from a single HepG2 cell whose phenotypic behavior we have observed via microscopic imaging before capture. In project avenue two, over the past year we have been attempting to add micro-RNA [miR] measurements to our ‘cue-signal-response’ paradigm, since over the past few years a significant role for miRs in modulating signaling protein levels has emerged. We have established a Luminex-based technique for multi-plexed dynamic measurement of putative key miRs in hepatocytic cells (primary and lines) following inflammatory cytokine treatment, for purpose of generating partial least-squares regression [PLSR] models capable of predicting apoptosis responses in manner analogous to (or complementary to) our earlier successes using PLSR modeling centered on kinase activity and phosphoprotein levels. We have demonstrated proof-of-concept of this new approach for Huh7 cells responding to permutations of TRAIL and Inf-g stimuli, based on time-courses of six miRs across 48 hours post-treatment. Interestingly, the miR measurement time-points seem to be the most important feature for predictive capability, rather than miR identities. In project avenue three, over the past year we have been endeavoring to incorporate multiple alternative phenotypic responses for cells in a given environment. Employing microvascular endothelial cells, we have by microscopic imaging followed hundreds of individual cells as they proliferate, migrate, or die in response to various combinations of VEGF and PF4. This represents an experimental instantiation of angiogenesis in inflammatory contexts, with VEGF being “pro-angiogenic” and PF4 being “anti-angiogenic” – in both cases influencing all three of the above phenotypic behaviors. We have found that transition probabilities among these three responses fall into an identifiable, small number of clusters, with the proportion of cells exhibiting a particular cluster being influenced by the environmental conditions. |
