Automatic detection of spatio-temporal signaling patterns in cell collectives.

Increasing experimental evidence points to the physiological importance of space-time correlations in signaling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of biomolecules between cells allows the collectives to perform more complex tasks and to better tackle environmental challenges. To capture this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signaling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We made a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells hyperstimulate intercellular ERK activity waves that are largely dependent on matrix metalloproteinase intercellular signaling. ARCOS is open-source and available as R and Python packages. It also includes a plugin for the napari image viewer to interactively quantify collective phenomena without prior programming experience.

Systems-level transcriptional regulation of Caenorhabditis elegans metabolism.

Metabolism is controlled to ensure organismal development and homeostasis. Several mechanisms regulate metabolism, including allosteric control and transcriptional regulation of metabolic enzymes and transporters. So far, metabolism regulation has mostly been described for individual genes and pathways, and the extent of transcriptional regulation of the entire metabolic network remains largely unknown. Here, we find that three-quarters of all metabolic genes are transcriptionally regulated in the nematode Caenorhabditis elegans. We find that many annotated metabolic pathways are coexpressed, and we use gene expression data and the iCEL1314 metabolic network model to define coregulated subpathways in an unbiased manner. Using a large gene expression compendium, we determine the conditions where subpathways exhibit strong coexpression. Finally, we develop "WormClust," a web application that enables a gene-by-gene query of genes to view their association with metabolic (sub)-pathways. Overall, this study sheds light on the ubiquity of transcriptional regulation of metabolism and provides a blueprint for similar studies in other organisms, including humans.

Spatiotemporal control of ERK pulse frequency coordinates fate decisions during mammary acinar morphogenesis.

The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility and proliferation. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated multicellular ERK waves emerge, correlating with high and low ERK pulse frequencies in outer surviving and inner dying cells, respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation display increased ERK pulse frequency and inner cell survival. Thus, fate decisions during acinar morphogenesis are coordinated by different spatiotemporal modalities of ERK pulse frequency.

Optogenetic actuator - ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics.

Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.

Mining of Single-Cell Signaling Time-Series for Dynamic Phenotypes with Clustering.

Fluorescent live cell time-lapse microscopy is steadily contributing to our better understanding of the relationship between cell signaling and fate. However, large volumes of time-series data generated in these experiments and the heterogenous nature of signaling responses due to cell-cell variability hinder the exploration of such datasets. The population averages insufficiently describe the dynamics, yet finding prototypic dynamic patterns that relate to different cell fates is difficult when mining thousands of time-series. Here we demonstrate a protocol where we identify such dynamic phenotypes in a population of PC-12 cells that respond to a range of sustained growth factor perturbations. We use Time-Course Inspector, a free R/Shiny web application to explore and cluster single-cell time-series.

Collective ERK/Akt activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival.

Cell death events continuously challenge epithelial barrier function yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of extracellular-signal-regulated kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

CODEX, a neural network approach to explore signaling dynamics landscapes.

Current studies of cell signaling dynamics that use live cell fluorescent biosensors routinely yield thousands of single-cell, heterogeneous, multi-dimensional trajectories. Typically, the extraction of relevant information from time series data relies on predefined, human-interpretable features. Without a priori knowledge of the system, the predefined features may fail to cover the entire spectrum of dynamics. Here we present CODEX, a data-driven approach based on convolutional neural networks (CNNs) that identifies patterns in time series. It does not require a priori information about the biological system and the insights into the data are built through explanations of the CNNs' predictions. CODEX provides several views of the data: visualization of all the single-cell trajectories in a low-dimensional space, identification of prototypic trajectories, and extraction of distinctive motifs. We demonstrate how CODEX can provide new insights into ERK and Akt signaling in response to various growth factors, and we recapitulate findings in p53 and TGFß-SMAD2 signaling.

Mining single-cell time-series datasets with Time Course Inspector.

SUMMARY: Thanks to recent advances in live cell imaging of biosensors, microscopy experiments can generate thousands of single-cell time-series. To identify sub-populations with distinct temporal behaviours that correspond to different cell fates, we developed Time Course Inspector (TCI)-a unique tool written in R/Shiny to combine time-series analysis with clustering. With TCI it is convenient to inspect time-series, plot different data views and remove outliers. TCI facilitates interactive exploration of various hierarchical clustering and cluster validation methods. We showcase TCI by analysing a single-cell signalling time-series dataset acquired using a fluorescent biosensor. AVAILABILITY AND IMPLEMENTATION: https://github.com/pertzlab/shiny-timecourse-inspector. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Temporal perturbation of ERK dynamics reveals network architecture of FGF2/MAPK signaling.

Stimulation of PC-12 cells with epidermal (EGF) versus nerve (NGF) growth factors (GFs) biases the distribution between transient and sustained single-cell ERK activity states, and between proliferation and differentiation fates within a cell population. We report that fibroblast GF (FGF2) evokes a distinct behavior that consists of a gradually changing population distribution of transient/sustained ERK signaling states in response to increasing inputs in a dose response. Temporally controlled GF perturbations of MAPK signaling dynamics applied using microfluidics reveal that this wider mix of ERK states emerges through the combination of an intracellular feedback, and competition of FGF2 binding to FGF receptors (FGFRs) and heparan sulfate proteoglycan (HSPG) co-receptors. We show that the latter experimental modality is instructive for model selection using a Bayesian parameter inference. Our results provide novel insights into how different receptor tyrosine kinase (RTK) systems differentially wire the MAPK network to fine-tune fate decisions at the cell population level.