PUBLICATION

DNA-based fluorescent probes of NOS2 activity in live brains

Authors
Veetil, A.T., Zou, J., Henderson, K.W., Jani, M.S., Shaik, S.M., Sisodia, S.S., Hale, M.E., Krishnan, Y.
ID
ZDB-PUB-200620-11
Date
2020
Source
Proceedings of the National Academy of Sciences of the United States of America   117(26): 14694-14702 (Journal)
Registered Authors
Hale, Melina
Keywords
live imaging, microglia, nitric oxide, nitric oxide synthase-2 (NOS2), phagosome
MeSH Terms
  • Animals
  • Brain/enzymology*
  • Brain/metabolism
  • Brain Chemistry
  • DNA/chemistry*
  • DNA/metabolism
  • Fluorescent Dyes/chemistry*
  • Fluorescent Dyes/metabolism
  • Gene Knockout Techniques
  • Mice
  • Microglia/chemistry
  • Microglia/enzymology
  • Microglia/metabolism
  • Microscopy, Fluorescence
  • Molecular Probes/chemistry
  • Molecular Probes/metabolism
  • Nitric Oxide Synthase Type II*/analysis
  • Nitric Oxide Synthase Type II*/chemistry
  • Nitric Oxide Synthase Type II*/metabolism
  • Phagosomes/chemistry
  • Phagosomes/metabolism
  • Zebrafish
PubMed
32554491 Full text @ Proc. Natl. Acad. Sci. USA
Abstract
Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping