Functional decoding of signaling dynamics in single immune cells

National Institute On Aging

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Abstract

We acquire 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 various 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, T cells, and ear fibroblasts. Our projects progressed substantially the past year, resulting in a research publication and three manuscripts in preparation. We have developed four novel knock-in mouse strains. 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 whole genome sequencing with PacBio long-read sequencing (Mahesh G et al, Sci Data 2024). 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. Simultaneous live cell imaging of RelA and c-Rel in naïve CD4 and CD8 T cells from the reporter mice showed unexpected importance of c-Rel subunit of NF-κB in gene activation after TCR stimulation (Aqdas M. et al. in preparation). By crossing the knock-in NF-κB reporter mice to a mouse model of Alzheimer's disease, we analyzed the changes in amyloid beta phagocytosis and clearance in microglia (Songkiatisak P. et al. in preparation). In another study (Oh Y. et al. in preparation), we characterized the altered responses of NF-κB in fibroblasts to pro-inflammatory cytokines under a wide range of temperatures from ambient to febrile temperature conditions.