A guide to ERK dynamics, part 1: mechanisms and models.

Extracellular signal-regulated kinase (ERK) has long been studied as a key driver of both essential cellular processes and disease. A persistent question has been how this single pathway is able to direct multiple cell behaviors, including growth, proliferation, and death. Modern biosensor studies have revealed that the temporal pattern of ERK activity is highly variable and heterogeneous, and critically, that these dynamic differences modulate cell fate. This two-part review discusses the current understanding of dynamic activity in the ERK pathway, how it regulates cellular decisions, and how these cell fates lead to tissue regulation and pathology. In part 1, we cover the optogenetic and live-cell imaging technologies that first revealed the dynamic nature of ERK, as well as current challenges in biosensor data analysis. We also discuss advances in mathematical models for the mechanisms of ERK dynamics, including receptor-level regulation, negative feedback, cooperativity, and paracrine signaling. While hurdles still remain, it is clear that higher temporal and spatial resolution provide mechanistic insights into pathway circuitry. Exciting new algorithms and advanced computational tools enable quantitative measurements of single-cell ERK activation, which in turn inform better models of pathway behavior. However, the fact that current models still cannot fully recapitulate the diversity of ERK responses calls for a deeper understanding of network structure and signal transduction in general.

A guide to ERK dynamics, part 2: downstream decoding.

Signaling by the extracellular signal-regulated kinase (ERK) pathway controls many cellular processes, including cell division, death, and differentiation. In this second installment of a two-part review, we address the question of how the ERK pathway exerts distinct and context-specific effects on multiple processes. We discuss how the dynamics of ERK activity induce selective changes in gene expression programs, with insights from both experiments and computational models. With a focus on single-cell biosensor-based studies, we summarize four major functional modes for ERK signaling in tissues: adjusting the size of cell populations, gradient-based patterning, wave propagation of morphological changes, and diversification of cellular gene expression states. These modes of operation are disrupted in cancer and other related diseases and represent potential targets for therapeutic intervention. By understanding the dynamic mechanisms involved in ERK signaling, there is potential for pharmacological strategies that not only simply inhibit ERK, but also restore functional activity patterns and improve disease outcomes.

Deciphering the History of ERK Activity from Fixed-Cell Immunofluorescence Measurements.

The Ras/ERK pathway drives cell proliferation and other oncogenic behaviors, and quantifying its activity in situ is of high interest in cancer diagnosis and therapy. Pathway activation is often assayed by measuring phosphorylated ERK. However, this form of measurement overlooks dynamic aspects of signaling that can only be observed over time. In this study, we combine a live, single-cell ERK biosensor approach with multiplexed immunofluorescence staining of downstream target proteins to ask how well immunostaining captures the dynamic history of ERK activity. Combining linear regression, machine learning, and differential equation models, we develop an interpretive framework for immunostains, in which Fra-1 and pRb levels imply long term activation of ERK signaling, while Egr-1 and c-Myc indicate recent activation. We show that this framework can distinguish different classes of ERK dynamics within a heterogeneous population, providing a tool for annotating ERK dynamics within fixed tissues.

Live-Cell Sender-Receiver Co-cultures for Quantitative Measurement of Paracrine Signaling Dynamics, Gene Expression, and Drug Response.

Paracrine signaling is a fundamental process regulating tissue development, repair, and pathogenesis of diseases such as cancer. Herein we describe a method for quantitatively measuring paracrine signaling dynamics, and resultant gene expression changes, in living cells using genetically encoded signaling reporters and fluorescently tagged gene loci. We discuss considerations for selecting paracrine "sender-receiver" cell pairs, appropriate reporters, the use of this system to ask diverse experimental questions and screen drugs blocking intracellular communication, data collection, and the use of computational approaches to model and interpret these experiments.

ERK signaling dynamics: Lights, camera, transduction.

Three-dimensional mammary epithelial acini are a model for understanding how microenvironment-driven signaling coordinates cell behavior and tissue morphogenesis. In this issue of Developmental Cell, Ender et al. use live-cell imaging to capture dynamic spatiotemporal patterns of ERK activity that instruct cell migration and survival fates in developing acini.

Entosis is induced by ultraviolet radiation.

Entosis is a cell death mechanism that is executed through neighbor cell ingestion and killing that occurs in cancer tissues and during development. Here, we identify JNK and p38 stress-activated kinase signaling as an inducer of entosis in cells exposed to ultraviolet (UV) radiation. Cells with high levels of stress signaling are ingested and killed by those with low levels, a result of heterogeneity arising within cell populations over time. In stressed cells, entosis occurs as part of mixed-cell death response with parallel induction of apoptosis and necrosis, and we find that inhibition of one form of cell death leads to increased rates of another. Together, these findings identify stress-activated kinase signaling as a new inducer of entosis and demonstrate cross talk between different forms of cell death that can occur in parallel in response to UV radiation.

Systems-Level Properties of EGFR-RAS-ERK Signaling Amplify Local Signals to Generate Dynamic Gene Expression Heterogeneity.

Intratumoral heterogeneity is associated with aggressive tumor behavior, therapy resistance, and poor patient outcomes. Such heterogeneity is thought to be dynamic, shifting over periods of minutes to hours in response to signaling inputs from the tumor microenvironment. However, models of this process have been inferred from indirect or post-hoc measurements of cell state, leaving the temporal details of signaling-driven heterogeneity undefined. Here, we developed a live-cell model system in which microenvironment-driven signaling dynamics can be directly observed and linked to variation in gene expression. Our analysis reveals that paracrine signaling between two cell types is sufficient to drive continual diversification of gene expression programs. This diversification emerges from systems-level properties of the EGFR-RAS-ERK signaling cascade, including intracellular amplification of amphiregulin-mediated paracrine signals and differential kinetic filtering by target genes including Fra-1, c-Myc, and Egr1. Our data enable more precise modeling of paracrine-driven transcriptional variation as a generator of gene expression heterogeneity. A record of this paper's transparent peer review process is included in the Supplemental Information.