Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression.

The Ras-ERK pathway controls cell proliferation and differentiation, whereas the PI3K-Akt pathway plays a role in the process of cell-cycle progression and cell survival. Both pathways are activated by many stimuli such as epidermal growth factor (EGF), and coordinately regulate each other through cross-talk. However, it remains unclear how cells accommodate the dynamics and interplay between the Ras-ERK and PI3K-Akt pathways to regulate cell-fate decisions, mainly because of the lack of good tools to visualize ERK and Akt activities simultaneously in live cells. Here, we developed a multiplexed fluorescence system for imaging ERK and Akt signaling and the cell-cycle status at the single cell level. Based on the principle of the kinase translocation reporter (KTR), we created Akt-FoxO3a-KTR, which shuttled between nucleus and cytoplasm in a manner regulated by Akt phosphorylation. To simultaneously measure ERK, Akt and the cell-cycle status, we generated a polycistronic vector expressing ERK-KTR, Akt-FoxO3a-KTR, a cell-cycle reporter and a nuclear reporter, and applied linear unmixing to these four images to remove spectral overlap among fluorescent proteins. The specificity and sensitivity of ERK-KTR and Akt-FoxO3a-KTR were characterized quantitatively. We examined the cellular heterogeneity of relationship between ERK and Akt activities under a basal or EGF-stimulated condition, and found that ERK and Akt were regulated in a highly cooperative and cell-cycle-dependent manner. Our study provides a useful tool for quantifying the dynamics among ERK and Akt activities and the cell cycle in a live cell, and for addressing the mechanisms underlying intrinsic resistance to molecularly targeted drugs.