Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes

Hirotaka Fujita, Ryota Oikawa, Mayu Hayakawa, Fumiaki Tomoike, Yasuaki Kimura, Hiroyuki Okuno, Yoshiki Hatashita, Carolina Fiallos Oliveros, Haruhiko Bito, Toshio Ohshima, Satoshi Tsuneda, Hiroshi Abe, Takafumi Inoue

Research output: Contribution to journalArticle

Abstract

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and IP3 receptor isoform 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF–FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

Original languageEnglish
JournalJournal of Biological Chemistry
Volume295
Issue number23
DOIs
Publication statusPublished - 2020 Apr

Keywords

  • Cellular dynamics
  • Fluorescence correlation spectroscopy (FCS)
  • Gene regulation
  • Hippocampus
  • Live-cell imaging
  • MRNA
  • Neuron
  • Reduction-triggered fluorescence (RETF)
  • RNA transport
  • Transcription

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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