Redefining Signaling Pathways with an Expanding Single-Cell Toolbox

Abstract

Genetically identical cells respond heterogeneously to uniform environmental stimuli. Consequently, investigating the signaling networks that control these cell responses using 'average' bulk cell measurements can obscure underlying mechanisms and misses information emerging from cell-to-cell variability. Here we review recent technological advances including live-cell fluorescence imaging-based approaches and microfluidic devices that enable measurements of signaling networks, dynamics, and responses in single cells. We discuss how these single-cell tools have uncovered novel mechanistic insights for canonical signaling pathways that control cell proliferation (ERK), DNA-damage responses (p53), and innate immune and stress responses (NF-κB). Future improvements in throughput and multiplexing, analytical pipelines, and in vivo applicability will all significantly expand the biological information gained from single-cell measurements of signaling pathways.

Figure 1. Overview of technologies developed to interrogate or modulate ERK activity in single cells

A) A FRET-based sensor undergoes a conformational change upon phosphorylation by ERK that is detected by a change in the ratio of donor-to-acceptor fluorescence emission intensity. B) Fluorescent KTRs translocate from the nucleus to the cytoplasm when phosphorylated by ERK. C) FIRE (Fra-1-based integrative reporter of ERK) is stabilized upon phosphorylation by ERK leading to an increase in fluorescence intensity. D) Light activates an optogenetic membrane localized ERK-activating signal (SOS_cat) to enable light-based control of ERK activation. FP: fluorescent protein. FHA1, Elk-1, FRA-1: substrate domains modulated by active ERK.

Figure 2. Regulatory signaling mechanisms discovered from single-cell data

A) ERK activity increases linearly with EGF concentration as measured in a cell population. Single-cell data showed that this increase is due to increased frequency of short pulses of ERK activity. B) p53 abundance increases linearly with UV intensity. Measuring both total and active tetramer abundance in single cells revealed that active tetramer abundance increases much more gradually than the total abundance, constraining the dose response. C) For NF-κB, single-cell data showed that the strongly dampened oscillations observed at the population level are due to loss of synchrony in oscillations following the first peak. While the intercellular oscillatory period varies, the intracellular period is relatively constant, and altogether the population produces stable and largely invariant NF-κB oscillation pattern. D) Population-averaged nuclear NF-κB abundance and that of its target transcripts are positively correlated but vary across cells. By measuring time courses of NF-κB translocation in single cells, it becomes apparent that the fold change in nuclear NF-κB – not its absolute abundance – more precisely determines the transcriptional output in individual cells. E) Nano-well experiments have shown that strong inflammatory signals released in a population of LPS-treated cells are dependent on paracrine signaling. F) Single-cell studies allow the investigation of how cell shape and cell microenvironment influence signaling responses, including the nuclear localization of NF-κB.