We report an optimized backbone for the rapid development of a highly sensitive intramolecular fluorescence resonance energy transfer (FRET) biosensor, which includes an optimized pair of fluorescent proteins and a long flexible linker ranging from 116 to 244 amino acids in length. With this backbone system, we developed FRET biosensors of PKA, ERK, JNK, EGFR, RSK, S6K, Akt, PKC, Ras, and Rac1.

The extracellular signal-regulated kinase (ERK) plays a central role in the signaling cascades of cell growth. Here, we show that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner. A fluorescence resonance energy transfer (FRET) biosensor revealed that stochastic ERK activity pulses fired spontaneously or propagated from adjacent cells. Frequency, but not amplitude, of ERK activity pulses exhibited a bell-shaped response to the cell density and correlated with cell proliferation rates. Consistently, synthetic ERK activity pulses generated by a light-switchable CRaf protein accelerated cell proliferation. A mathematical model clarified that 80% and 20% of ERK activity pulses are generated by the noise and cell-to-cell propagation, respectively. Finally, RNA sequencing analysis of cells subjected to the synthetic ERK activity pulses suggested the involvement of serum responsive factor (SRF) transcription factors in the gene expression driven by the ERK activity pulses.

Extracellular signal-regulated kinase (ERK) is a key effector of many growth signalling pathways. In this study, we visualise epidermal ERK activity in living mice using an ERK FRET biosensor. Under steady-state conditions, the epidermis occasionally revealed bursts of ERK activation patterns where ERK activity radially propagated from cell to cell. The frequency of this spatial propagation of radial ERK activity distribution (SPREAD) correlated with the rate of epidermal cell division. SPREADs and proliferation were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in a manner dependent on EGF receptors and their cognate ligands. At the wounded skin, ERK activation propagated as trigger wave in parallel to the wound edge, suggesting that ERK activation propagation can be superimposed. Furthermore, by visualising the cell cycle, we found that SPREADs were associated with G2/M cell cycle progression. Our results provide new insights into how cell proliferation and transient ERK activity are synchronised in a living tissue.DOI: http://dx.doi.org/10.7554/eLife.05178.001

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Adhesion molecules can initiate intracellular signaling. Engagement of CD44 either by its natural ligand hyaluronan or a speci¢c antibody on a cell line induced tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which then associated with phosphatidylinositol 3-kinase (PI3K) and activated mitogen-activated protein kinase at its downstream. However, the introduction of dominant negative Rho into the cells inhibited the CD44-stimulated FAK phosphorylation. Cells expressing CD44 were signi¢cantly resistant to etoposide-induced apoptosis. This anti-apoptotic e¡ect was cancelled by the inhibition of either Rho, FAK or PI3K. These results may indicate a signaling pathway from CD44 to mediate the resistance against drug-induced apoptosis in cancer cells. ß