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.
Recently, researchers have developed a novel technology to generate single cell reporters for kinase activity. Their approach is based on the concept of converting phosphorylation into a nucleocytoplasmic shuttling event. In fact, there are numerous examples of phosphorylation-regulated nucleo-cytoplasmic translocation in naturally occurring proteins. The scientists hypothesized that by understanding this phenomena they 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, they defined a set of rules that they can use to engineer single cell kinase activity reporters. They named these reporters Kinase Translocation Reporters (KTR). In addition, they showed that KTR technology is generalizable by implementing KTR sensors for JNK, p38, ERK and PKA, thus covering different types of kinases. The researchers also used this technology to show that multiplexing capabilities go beyond any current method. In particular, they measured JNK, p38 and ERK activities simultaneously in live single cells.
This technology opens the possibility of analyzing multiple signaling networks, cell cycle and a broad range of kinase-mediated processes simultaneously in live single cells.