Following cytokine stimulation, NEMO is recruited to polyubiquitin scaffolds associated with cytokine-ligated receptor complexes where NEMO-interacting IκB kinases (IKKs) are activated through induced proximity with other signaling mediators ( Clark et al., 2013 Ea et al., 2006 Hayden and Ghosh, 2008 Pabon et al., 2019 Poyet et al., 2000). Upstream kinase activation by the NF-κB essential modulator protein (NEMO, also known as IKKɣ) is a necessary step in regulation of the classical NF-κB signaling cascade ( Liu et al., 2012). Following activation of the pathway by inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor (TNF) among others, a series of intracellular signaling events transduce the NF-κB signal. The nuclear factor (NF)-κB signal transduction pathway is a master regulator of inflammatory responses to injury and infection ( Zhang et al., 2017b). Accurate detection and quantification of biological puncta is necessary to examine their roles in regulating cellular behaviors, and computational analysis is often the rate-limiting step of experimental pipelines. However, most live-cell imaging approaches use fluorescent biosensors and fusion proteins that report within large subcellular compartments, such as the cytoplasm or nucleus, and quantification of punctate structures is often limited to fixed-cell and low-throughput applications. Spatiotemporal dynamics of signaling proteins, quantified in single cells by live-cell imaging, have revealed mechanistic insights into signal–response relationships in signal transduction networks in addition to sources of cell-to-cell variability ( Gaudet and Miller-Jensen, 2016 Xu et al., 2005 Zhang et al., 2017a).
Processes of the central dogma, such as active transcription, single mRNA transcripts, sites of active protein translation and other regulatory multi-protein assemblies can be observed by fluorescence microscopy as punctate structures within the cell ( Femino et al., 1998 Larson et al., 2005 Lyon et al., 2019 Raj and van Oudenaarden, 2009 Tanenbaum et al., 2014 Wang et al., 2016). Quantitative imaging of single cells enables measurement with subcellular resolution of dynamic biological processes that regulate critical cellular behaviors.
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