Visualization of dynamics of single endogenous mRNA labeled in live mouse - PubMed (original) (raw)
Visualization of dynamics of single endogenous mRNA labeled in live mouse
Hye Yoon Park et al. Science. 2014.
Abstract
The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.
Figures
Fig. 1
Labeled endogenous mRNA in MCP×MBS mouse. (A) Schematic for in vivo RNA labeling. NLS, nuclear localization sequence; HA, hemagglutinin; pol II, RNA polymerase II. (B and C) Single-particle tracking of GFP-labeled β-actin mRNP in primary MEF (B) and hippocampal neuron (C) from mouse. Track classifications: red, stationary; green, corralled; blue, diffusive; purple, directed motion (see supplementary text). N, nucleus; C, cytoplasm; S, soma. Scale bars, 10 μm. (D and E) Intensity histograms of β-actin mRNPs tracked in primary MEFs (D) and neurons (E). Red curves show one- and three-component Gaussian fits for (D) and (E), respectively. (F and G) Fraction of stationary, corralled, diffusive, and directed β-actin mRNPs in primary MEFs (F) and neurons (G). Error bars denote SEM (n = 6 cells).
Fig. 2
Activity-dependent dynamics of β-actin mRNP in neurons from MCP×MBS mouse. (A and B) Examples of β-actin mRNP merge (A) and split (B) events. From top to bottom: initial images of the particles, kymographs during a 12-s period, and final images. (C) Ratio of split to merge events increased after KCl depolarization (n = 11 neurons from three cultures, 14 to 16 days in vitro; P < 0.05 by t test). (D) CA1 region in acute hippocampal slice. Scale bar, 50 μm. (E) Soma layer of CA1 neurons. Panels show maximum projections of x-y, y-z, and x-z planes. Scale bar, 10 μm. (F) Transcription of β-actin gene with KCl depolarization. Arrowheads denote β-actin transcription sites. Scale bar, 5 μm. (G) Nascent mRNA per transcription site increased after depolarization (n = 86 sites, brain slices from 3 mice; P < 0.001 by pairwise t test). Error bars denote SEM.
Comment in
- Molecular biology. mRNA, live and unmasked.
Akbalik G, Schuman EM. Akbalik G, et al. Science. 2014 Jan 24;343(6169):375-6. doi: 10.1126/science.1249623. Science. 2014. PMID: 24458628 No abstract available. - Synaptic plasticity: unmasked: dendritic mRNA dynamics.
Whalley K. Whalley K. Nat Rev Neurosci. 2014 Mar;15(3):138-9. doi: 10.1038/nrn3697. Epub 2014 Feb 12. Nat Rev Neurosci. 2014. PMID: 24518418 No abstract available.
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