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*

*この研究の対応する著者

研究成果: Article査読

2 被引用数 (Scopus)

抄録

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.

本文言語English
ジャーナルJournal of Biological Chemistry
295
23
DOI
出版ステータスPublished - 2020 4月

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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