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. 2017 Jun 1;28(11):1412-1417.
doi: 10.1091/mbc.E16-07-0497. Epub 2017 Apr 12.

Components of RNA granules affect their localization and dynamics in neuronal dendrites

Kazuhiko Mitsumori  1 , Yosuke Takei  2   3 , Nobutaka Hirokawa  2   4
Affiliations

Components of RNA granules affect their localization and dynamics in neuronal dendrites

Kazuhiko Mitsumori et al. Mol Biol Cell. .

Abstract

In neurons, RNA transport is important for local protein synthesis. mRNAs are transported along dendrites as large RNA granules. The localization and dynamics of Puralpha and Staufen1 (Stau1), major components of RNA transport granules, were investigated in cultured hippocampal neurons. Puralpha-positive granules were localized in both the shafts and spines of dendrites. In contrast, Stau1-positive granules tended to be localized mainly in dendritic shafts. More than 90% of Puralpha-positive granules were positive for Stau1 in immature dendrites, while only half were positive in mature dendrites. Stau1-negative Puralpha granules tended to be stationary with fewer anterograde and retrograde movements than Stau1-positive Puralpha granules. After metabotropic glutamate receptor 5 activation, Stau1-positive granules remained in the dendritic shafts, while Puralpha granules translocated from the shaft to the spine. The translocation of Puralpha granules was dependent on myosin Va, an actin-based molecular motor protein. Collectively our findings suggest the possibility that the loss of Stau1 in Puralpha-positive RNA granules might promote their activity-dependent translocation into dendritic spines, which could underlie the regulation of protein synthesis in synapses.

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Figures

FIGURE 1:
FIGURE 1:
(A) Mature hippocampal neurons were immunostained with anti-Puralpha and anti–PSD-95 antibodies. White arrows indicate the colocalization of Puralpha and PSD-95 clusters. (B) Neurons were transfected with TagRFP and immunostained with anti-Puralpha antibody. Note that some Puralpha clusters are localized in dendritic spines (arrows). (C) Neurons were immunostained with anti-Stau1 and anti–PSD-95. (D) Neurons were transfected with TagRFP and immunostained with anti-Stau1 antibody. (E) Quantification. Error bars indicate SEM; 10–15 cells from three mice were examined. (F) Mature hippocampal neurons were transfected with YFP-Puralpha and immunostained with anti–PSD-95. White arrows indicate the colocalization of Puralpha and PSD-95 clusters. (G) TagRFP was coexpressed with YFP-Puralpha. Some Puralpha clusters localize to dendritic spines (arrows). (H) Neurons were transfected with Stau1-GFP and were subsequently immunostained with anti–PSD-95. (I) TagRFP is coexpressed with Stau1-GFP. (J) Quantification. Error bars indicate SEM; 20–27 cells from three mice were examined. ***, p < 0.001; Student’s t test. Scale bars: 5 μm.
FIGURE 2:
FIGURE 2:
(A) In immature dendrites (8 DIV), almost all of the TagRFP-Puralpha clusters colocalized with Stau1-GFP clusters (white arrows). (B) In mature dendrites (15 DIV), YFP-Puralpha clusters only partially colocalized with Stau1-TagRFP clusters (white arrows). p < 0.001; Student’s t test. Scale bars: 5 μm. (C–E) In immature (C) or mature (D) dendrites, TagRFP-Puralpha clusters were cotransported with Stau1-GFP clusters (white arrows). Asterisks indicate initial position. (E) Puralpha-positive granules (asterisks) were classified into Stau1-positive/Puralpha-positive granules (solid arrowheads) and Stau1-negative/Puralpha-positive clusters (open arrowheads). Scale bars: 2 μm.
FIGURE 3:
FIGURE 3:
(A) Time-lapse recording of YFP-Puralpha clusters after DHPG treatment reveals that YFP-Puralpha accumulates within spines. Scale bar: 10 μm. (B) The average cluster index is increased 30 min after treatment with 100 μM DHPG (22 clusters from 12 neurons from two mice were examined). The DHPG-dependent spine accumulation is abolished by MPEP, an mGluR5 antagonist (18 clusters from 11 neurons from two mice were examined). For the control, 22 clusters from 12 neurons from two mice were examined. Error bars indicate SEM. ***, p < 0.001; Student’s t test. (C) Time-lapse recording after DHPG treatment reveals that Stau1-GFP clusters remained in dendritic shafts. Scale bar: 10 μm. (D) The average cluster index is not increased 30 min after treatment with DHPG (12 clusters from five neurons from two mice were examined). For control, 12 clusters from six neurons from two mice were examined. Error bars indicate SEM. (E) Forty-eight hours after transfection with miR vectors, neurons (15 DIV) were treated with DHPG for 1 h, fixed, and stained with anti-Puralpha antibodies. Scale bars: 10 μm. (F) Statistical analysis of E. Percentage of spines containing endogenous Puralpha clusters was increased by DHPG treatment and was decreased by myosin Va silencing. Error bars represent SEM. **, p < 0.01; Student’s t test. (G) Schematic model. Puralpha clusters are localized in both dendritic spines and shafts, whereas Stau1 clusters are present only in dendritic shafts. Some of these clusters are transported along dendrites by microtubule-based molecular motors (KIF5). Stau1-negative clusters are preferentially translocated to dendritic spines by an actin-based molecular motor (myosin Va). This oriented translocation involves activation of the mGluR5 pathway.
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