Molecular Systems Biology

Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain‐of‐function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high‐content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal‐developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine‐rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase α (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine‐related pathways significantly inhibited neurite formation.

Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single‐cell resolution. However, probing signaling in epithelial tissues using cytometry‐based single‐cell analysis has been confounded by the necessity of single‐cell dissociation, where disrupting cell‐to‐cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue—DISSECT) that preserves native signaling for Cytometry Time‐of‐Flight (CyTOF) and fluorescent flow cytometry applications. A 21‐plex CyTOF analysis encompassing core signaling and cell‐identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor‐alpha (TNF‐α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF‐α‐induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p‐ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact‐dependent survival. Our novel single‐cell approach can broadly be applied, using both CyTOF and multi‐parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.