Functional decoding of signaling dynamics in single immune cells

National Institute On Aging

Investigators

Linked publications & trials

Abstract

We acquired real time dynamics data from a sensitive laser scanning microscope system with a built-in long-term tissue incubation capability. The NF-kappaB subunits RelA and c-Rel are transcriptional regulators that mediate the responses of cells to stimulation of various receptors by different pathogenic stimuli. We investigated how the patterns of RelA and c-Rel signaling dynamics differ in multiple primary cell types including brain-resident microglia, bone marrow derived macrophages, and ear fibroblasts. Despite the COVID-19 variant surges and fur mite infestation encountered in our vivarium, our projects progressed substantially the past year, resulting in a research manuscript currently under revision for the journal Cell Reports. We have developed four novel knock-in mouse strains since 2016. Each strain expresses a fluorescent NF-kappaB subunit fusion from the native genomic locus, by CRISPR-Cas genome engineering. We verified correct integration of the targeting constructs by Southern blotting, PCR and sequencing. We have also generated homozygous colonies of double knock-in mice where both RelA and c-Rel genes are endogenously labeled with a fluorescent protein. Overcoming hurdles in simultaneous live cell imaging of RelA and c-Rel in cells from the reporter mice, we found that how the signaling activities of distinct dimers of NF-B are cross-related with each other to achieve selective gene expression programs in macrophages and microglia. Our analysis also revealed that the quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells of the same type, and shift toward c-Rel in microglia from aged brains. We also identified an unexpected depletion of nuclear RelA:c-Rel heterodimers in stimulated cells. Quantitative same-cell measurements in these mice revealed a trajectory of subunit expression in several immune lineages, with a surprising downregulation at key cell maturation stages. These data begin to reveal the power of these reporters in gaining deeper insights to NF-B-linked biology, with the spectral complementarity of the labeled NF-B proteins enabling diverse applications from single-molecule analyses to in situ identification of cells in active inflammatory states.