Observing kinase activity in live single cells [with video]
Single cells are intrinsically noisy and need to be analyzed individually. We describe a novel generalizable method to measure multiple kinase activities simultaneously in live single cells. Our technology opens the exciting possibility of dynamically analyzing multiple signaling networks, as well as cell cycle and a wide range of kinase mediated processes simultaneously at the single-cell level.
HFSP Long-Term Fellow Sergi Regot and colleaguesauthored on Fri, 20 June 2014
Ongoing efforts have shown that multicellular systems are best understood as a combination of heterogeneous single cell behaviors. Intrinsic noise generates cell-to-cell variation that can be critical for cellular survival, development and differentiation. In response to changing environments, cells also generate complex signaling dynamics that encode relevant information for gene expression, proliferation or stress responses. Indeed, bulk population dynamics are often qualitatively different from single cell behaviors. As a result, live-cell microscopy has acquired a central role to study single cell biology. Dynamic single cell reporters are essential for live-cell microscopy. However, the number and type of molecular events that can be dynamically monitored in an individual cell is small. Such reporters have led to the successful measurement of metabolic state, transcription factor localization and even protein activities in live single cells. In the latter category, kinase activities are of particular interest. Kinases are known to regulate multiple and diverse biological functions, including the cell cycle, the innate immune response, development and cell differentiation.
In this study, we have recently developed a novel technology to generate single cell reporters for kinase activity. Our approach is based on the concept of converting phosphorylation into a nucleocytoplasmic shuttling event. In fact, there are numerous examples of phosphorylation-regulated nucleocytoplasmic translocation in naturally occurring proteins. We hypothesized that by understanding this phenomena we could synthetically engineer single color kinase reporters for single cells. Therefore, after exploring the sequence space using the JNK (c-Jun N terminal Kinase) substrate c-Jun, we defined a set of rules that we can now use to engineer single cell kinase activity reporters. We named these reporters Kinase Translocation Reporters (KTR) (Figure 1). In addition, we showed that KTR technology is generalizable by implementing KTR sensors for JNK, p38, ERK and PKA, thus covering different types of kinases. We then used this technology to show that multiplexing capabilities go beyond any current method. In particular, we measured JNK, p38 and ERK activities simultaneously in live single cells. This study opens the possibility of analyzing multiple signaling networks, cell cycle and a broad range of kinase-mediated processes simultaneously in live single cells.
High-Sensitivity Measurements of Multiple Kinase Activities in Live Single Cells. Sergi Regot, Jacob J. Hughey, Bryce T. Bajar, Silvia Carrasco, Markus W. Covert. Cell 157(7): 1724-1734.