1. Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes
- Author
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Yasuaki Kimura, Hiroshi Abe, Ryota Oikawa, Haruhiko Bito, Fumiaki Tomoike, Takafumi Inoue, Toshio Ohshima, Carolina Fiallos Oliveros, Mayu Hayakawa, Hirotaka Fujita, Satoshi Tsuneda, Yoshiki Hatashita, and Hiroyuki Okuno
- Subjects
0301 basic medicine ,RNA localization ,RNA transport ,Fluorescence correlation spectroscopy ,Nerve Tissue Proteins ,Biochemistry ,03 medical and health sciences ,Live cell imaging ,Transcription (biology) ,medicine ,Animals ,Inositol 1,4,5-Trisphosphate Receptors ,RNA, Messenger ,Rats, Wistar ,Molecular Biology ,Cells, Cultured ,Fluorescent Dyes ,Neurons ,Messenger RNA ,030102 biochemistry & molecular biology ,Chemistry ,Optical Imaging ,RNA ,Cell Biology ,Rats ,Cytoskeletal Proteins ,030104 developmental biology ,medicine.anatomical_structure ,Spectrometry, Fluorescence ,Biophysics ,Neuron ,Molecular Biophysics - 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 inositol 1,4,5-trisphosphate receptor type 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.
- Published
- 2019