| Signal transduction networks, comprised of interacting signaling pathways, control diverse cellular processes, and their chronic activation contributes to the progression of various diseases, such as cancer, diabetes, and immune disorders. In turn, signaling pathways are controlled by external cues (stimuli) present in the cell’s microenvironment. Signaling networks have been widely studied because of their importance in physiological processes. Quantitative imaging of live cells is providing unprecedented insights into signal transduction mechanisms for widely studied cellular responses like cell adhesion and migration. It has long been appreciated that spatiotemporal dynamics of cell migration are under the control of intracellular signaling pathways, yet the mechanisms by which signaling processes affect those behaviors remain unclear. We have developed analytical methods for relating parallel live-cell microscopy measurements of cell migration dynamics to the intracellular signaling processes that govern them. In one example of this approach, we used total internal reflection fluorescence (TIRF) microscopy to visualize spatiotemporal hot spots of signaling through the phosphoinositide 3-kinase (PI3K) pathway, which were found to coincide with localized cell protrusion and endure with characteristic lifetimes that correspond to those of cell migration persistence. Moreover, distant hot spots are found to be dynamically and stochastically coupled, and a PI3K-dependent mechanism was elucidated by which fibroblasts reorient the directionality of migration. In conjunction with our quantitative imaging approach, we have also utilized poly(vinylmethylsiloxane) networks, which are novel TIRF-compatible, chemically and mechanically tunable substrata for live-cell adhesion and migration studies. Studies on these substrates indicated that the synergy sequence of fibronectin (PHSRN) in addition to the RGD tri-peptide motif promotes more productive cell migration without markedly enhancing cell adhesion strength, compared to RGD alone.;In a different application of live-cell microscopy and image analysis, we investigated signaling through the extracellular signal-regulated kinase (ERK) pathway at the single-cell level, with the goal of parsing two prominent aspects of the signal transduction mechanism: the kinetics of pathway activation, which is known to be subject to negative feedback regulation, and compartmentalization of signaling components. ERK catalytic activity and nuclear translocation were measured simultaneously in live cells using a FRET-based ERK activity reporter (EKAR) probe and a mCherry-ERK2 fusion construct, respectively. We found that PDGF-stimulated ERK activation kinetics in nucleus and cytosol are distinct and strikingly different from those of ERK nuclear localization, observations that we reconciled using a newly developed mathematical model. Our analysis reveals a new conceptual model in which ERK interactions with nuclear substrates have a dramatic buffering effect, which shapes the apparent adaptation of the pathway.;These examples highlight the application of live-cell fluorescence microscopy as a versatile approach for studying spatiotemporal dynamics, stochasticity, and cell-to-cell heterogeneity of intracellular processes. |
