Real-time single-cell biology

Advances in imaging technologies have opened a new window on the dynamic biochemical events occurring in individual living cells. Automated microscopy allows populations of hundreds of cells to be observed simultaneously, while advances in fluorescent biosensors allow many signaling pathways to be tracked in real time. This technology has revealed two surprising facets of cell signaling. First, signaling events are unexpectedly dynamic, even under steady-state conditions. Second, even genetically identical cells can vary widely in their response to stimuli or drugs. Together, these findings indicate that there are still substantial gaps in our understanding of how many signaling pathways work and how they are interpreted by the cell.

Information flow in single cells

Our lab uses single-cell technologies to develop a more accurate mechanistic model of key signaling pathways regulating cell proliferation and survival. Pathways including ERK, Akt, mTOR, AMPK, and Hippo are activated by factors in the cellular microenvironment (including growth factors, nutrients, extracellular matrix contacts, and cell-cell interactions). Through the phosphorylation of numerous targets, this core signaling network controls central aspects of cellular behavior such as metabolic flux, growth, and DNA replication, which in turn determine cell phenotypes such as proliferation, apoptosis, and autophagy. A long-standing challenge in cell biology has been to understand how this limited set of kinase/transcription pathways can achieve specific control of cell fate in response to the diverse set of inputs integrated by the network. A potential answer to this question is suggested by the highly dynamic and individualized single-cell dynamics of ERK and other components of the core network, including mTOR, Akt, and AMPK. Such dynamic patterns are capable of carrying a large quantity of information and, as we have demonstrated, are capable of determining cell fate. We are exploring the hypothesis that 1) the unique pattern of signaling dynamics within each cell encodes the integrated status of multiple aspects of the microenvironment and that 2) differential cell fates are be specified by decoding certain patterns of core pathway activity.

Dynamic heterogeneity and drug responses

Targeted signaling inhibitors represent the forefront of modern cancer therapy, but most show limited long-term efficacy following initially promising responses, because of the ability of cancer cells to adapt to the loss of a single signaling pathway. Recent studies have found greatly increased efficacy when such inhibitors are administered intermittently or in a specific sequence with other drugs. However, the space of possible treatment regimens is large and cannot be explored practically using experiments alone. We are using our live-cell reporters to profile both acute and long-term signaling and metabolic behavior in response to targeted inhibitor treatments, with the goal of developing potential treatment regimes that direct the cellular signaling and metabolic state toward a target cell fate (e.g. cell death). This analysis takes into account the heterogeneity of states within the population and considers not only whether the majority of cells are induced to take on the target fate, but also whether undesirable secondary states (such as
rapid proliferation or differentiation into drug-resistant states) are effectively avoided.

Expanding the single-cell repertoire

The ability of live-cell microscopy to reveal signaling dynamics is still limited to a handful of pathways for which effective reporters have been developed. We are working to develop new and generalizeable strategies for reporter construction.