Disentangling Pro-mitotic Signaling during Cell Cycle Progression using Time-Resolved Single-Cell Imaging.

Cells rely on input from extracellular growth factors to control their proliferation during development and adult homeostasis. Such mitogenic inputs are transmitted through multiple signaling pathways that synergize to precisely regulate cell cycle entry and progression. Although the architecture of these signaling networks has been characterized in molecular detail, their relative contribution, especially at later cell cycle stages, remains largely unexplored. By combining quantitative time-resolved measurements of fluorescent reporters in untransformed human cells with targeted pharmacological inhibitors and statistical analysis, we quantify epidermal growth factor (EGF)-induced signal processing in individual cells over time and dissect the dynamic contribution of downstream pathways. We define signaling features that encode information about extracellular ligand concentrations and critical time windows for inducing cell cycle transitions. We show that both extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) activity are necessary for initial cell cycle entry, whereas only PI3K affects the duration of S phase at later stages of mitogenic signaling. • The contribution of EGF-induced signaling pathways is decomposed in single cells • Critical dynamic features are identified by information theory • ERK and PI3K activity are necessary for initial cell cycle entry • PI3K is necessary later to adjust the metabolism of the cell during replication Combining quantitative live-cell microscopy and single-cell sequencing with pharmacological perturbation, Benary et al. disentangle the dynamic requirements of EGF-induced MAPK and PI3K activity during mitogenic signaling. They corroborate that both pathways are necessary to induce S-phase entry and show that only PI3K is needed to drive replication by metabolic adjustments. [ABSTRACT FROM AUTHOR]