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. While the architecture of these signaling networks has been characterized in molecular detail, their relative contribution especially at later cell cycle stages remain largely unexplored. By combining quantitative time-resolved measurements of fluorescent reporters in untransformed human cells with targeted pharmacological inhibitors and statistical analysis, we quantified EGF-induced signal processing in individual cells over time and decomposed 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 ERK and PI3K activity is necessary for initial cell cycle entry, while only PI3K affect the duration of S-phase at later stages of mitogenic signaling. MCF10A cells stimulated cells with EGF and inhibited MEK and PI3K activity 15 h later. We then employed single cell RNA sequencing (scRNA-seq) to determine expression profiles of individual cells 20 h and 30 h after addition of the growth factor. The different conditions were multiplexed in one sample. Each sample resembles one biological replicate.