Article Figures & Data
Figures
- Figure 1.
A–E, Dendritic localization of P-body-like structures in rat hippocampal and hypothalamic neurons. In vitro cultured rat hypothalamic (B–D) or hippocampal (A) neurons were used for immunofluorescence analysis to study the distribution of several P-body markers. In A–D, scale bars represent 10 μm. The insets show enlargements of indicated regions (4×). A, Costaining of hippocampal neurons with anti-GW182 (bottom) and anti-PSD95 (middle; a marker for synapses) antibodies. GW182-positive granule present close to PSD95 positive synaptic site is marked by an arrow. Overlay (colored panel): GW182 staining is in green and PSD95 in red. DIC image of the same dendrite is shown in the top. B, Costaining of hypothalamic neurons with anti-Dcp1a (top) and anti-Ago2 (middle). Overlay (bottom): Dcp1a staining in green, Ago2 in red. C, Foci that are stained with anti-Dcp1a serum (left) were also labeled with anti-RCK/p54 antibodies (middle) in processes of cultured hypothalamic neurons. Overlay (right): Dcp1a is in green and rck/p54 is in red. D, Anti-Dcp1a antibody (left) labels foci in dendrites of rat hypothalamic neurons. Neurons were costained for neuronal dendritic marker MAP2 (middle). On the overlay (right) panel, Dcp1a is in green and the MAP2 staining is in red. Nuclei are stained with DAPI (in blue). E, Western blot reveal the presence of Dcp1a in crude and purified synaptosomal fractions prepared from the mouse brain extract.
- Figure 2.
Localization of miRNAs and their mRNA targets in dlP-bodies. A, B, Colocalization of miRNAs and GW182 in dlP-bodies of hypothalamic (A) and hippocampal (B) neurons. miRNAs let-7 (A) or miR-128 (B) were detected by in situ hybridization (top). Staining with anti-GW182 antibody is shown in middle panels. Overlay pictures are shown in bottom, with staining for miRNA in red and for GW182 in green. C, Hypothalamic neurons were cotransfected with plasmids expressing CFP-Ago2 (middle) and a Renilla luciferase (RL) mRNA reporter with three let-7 binding sites in its 3′ UTR (RL-3xB; top). In situ hybridization, using probes complementary to RL mRNA, detected the mRNA (red) in foci that are also positive for CFP-Ago2 (green; bottom). Scale bar, 10 μm. The insets show 4× enlargements of indicated areas. DAPI was used for staining the nuclei.
- Figure 3.
dlP-bodies contain translational repressor proteins. A, Anti-ZBP1 (top) and anti-GW182 (middle) antibody were used for detection of endogenous proteins in hippocampal neurons. On the overlay (bottom) panel, ZBP1 is in red, and GW182 in green. Colocalizations in panel (A–D) are marked by arrowheads. Scale bars, 10 μm. The insets show 4× enlargements of indicated regions. DAPI was used for staining the nuclei. B, Hypothalamic neurons transfected with CFP-Dcp1a (top) and YFP-ZBP1 (middle) show colocalization of CFP and YFP signals in discrete foci. Overlay (lower) panel shows colocalization of CFP-Dcp1 (red) with YFP-ZBP1 (green). C, GFP-tagged FMRP (middle) colocalizes with the endogenous Dcp1a (top) in dlP-bodies of hypothalamic neurons transfected with the GFP-FMRP expression plasmid. Overlay (bottom) panel shows colocalization of Dcp1a (red) with GFP-FMRP (green). D, Localization of SMN and Dcp1a proteins in hypothalamic neurons. Neurons expressing SMN-RFP (middle) were labeled with anti-Dcp1a (top). In overlay panel (bottom) Dcp1a is in green and SMN-RFP in red. E, ZBP1 and RCK/p54 coimmunoprecipitate with each other in protein extracts prepared from purified synaptosomes. Input represents 10% of the total extract used for each immunoprecipitation reaction. Rabbit polyclonal antibody against GRP78 protein was used in control immunoprecipitation.
- Figure 4.
dlP-bodies rarely contain the exonuclease Xrn1, but contain rRNA. A, Localization of Xrn1 (middle panel) and Dcp1a (top) in hypothalamic neurons transfected with a GFP-Dcp1a expression plasmid. The overlay (bottom) shows Xrn1 staining in red, GFP-Dcp1a in green and nuclei, stained with DAPI, in blue. A rabbit anti-Xrn1 antibody was used for detection of endogenous Xrn1. Insets (2× enlargements of the selected areas) show localization of both proteins in soma (left insets) and dendrites (right insets). B, Colocalization of rRNA (staining with Y10B mAb in top; red signal in the overlay panel) with anti-GW182 antibodies (middle; green in the overlay) in hippocampal neurons. Insets show 4× enlargements of the selected areas. C, D, Localization of rRNA (C) or Xrn1(D) in HeLa cells (top; red in the overlay panel) transfected with GFP-Dcp1a (middle panels; green in the overlay). Nuclei are stained with DAPI (blue in the overlay). Scale bars: 10 μm (in A, B); 5 μm (in C, D).
- Figure 5.
P-body components show directed and motorized movements along dendrites of rat neurons. Hypothalamic neurons transfected with GFP-Ago2 (A), GFP-Dcp1a (B) or GFP-SMN (C) were analyzed by time-lapse microscopy. Insets show enlargements (5× for GFPAgo2, 60× for GFP-Dcp1a, and 11× for GFP-SMN) of pictures taken at 250 ms intervals. Asterisks indicate initial position of foci, and arrows indicate their position at the indicated time.
- Figure 6.
A, B, Treatment with NMDA and other synaptic activators relocates dlP-bodies to distant parts of dendrites of rat neuron. Localization of GFP-Ago2 (A) and GFP-Dcp1a (B) positive foci in hypothalamic neurons either untreated (Ctrl) or after 15 min treatment with 30 μm NMDA. C, Quantification of the distance between dlP-bodies and the cell body. GFP-Ago2 (dark gray) and GFP-Dcp1a (light gray) in untreated neurons and in neurons treated with NMDA for 5 or 15 min. The numbers represent the maximum distance from cell soma in which GFP-Ago2 or GFP-Dcp1a foci were detectable. D, Distribution of GFP-Ago2 positive foci along dendrites before and after 15 min NMDA treatment (n = 11). E, Quantification of dendritic distribution of GFP-Dcp1a after 15 min treatment with different glutamatergic receptor agonists: NMDA (30 μm), kainate (50 μm) or DHPG (50 μm), or BDNF (50 mg/ml) and KCl (50 mm). When indicated, specific antagonists of NMDA and kainate, MK-801 (10 μm) and DNQX (50 μm), respectively, were added to the culture medium. The numbers the maximum distance from cell soma in which GFP-Ago2 or GFP-Dcp1a foci were detectable after different treatments (n = 14).
- Figure 7.
Synaptic stimulation modifies the exchange rates of of dlP-bodies components and modifies their composition. Hypothalamic neurons and HeLa cells expressing GFP-hDcp1a (A–C) or GFP-Ago2 (E–G) were analyzed by FRAP. Specific foci were bleached after 30 s recording, and fluorescent signals were recorded over time. Images of individual cells and enlargement of the bleached foci is shown. The graphs show recovery curves against time for GFPhDcp1a foci (D) or Ago2 foci (H). I, FRAP analysis of GFP-hDcp1 in dendrites before (blue) or after stimulation with 30 μm NMDA (red). J, Hypothalamic neurons were stimulated (30 μm NMDA for 15 min), and analyzed by immunofluorescence with antibodies against Ago2 and hDcp1a. K, Quantification of the fraction of Dcp1a foci that contained Ago2.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
- supplemental material - Supplemental Figure 3
- supplemental material - Supplemental Figure 4
- supplemental material - Supplemental Figure 5
- supplemental material - Supplemental Figure 6
- supplemental material - Supplemental Figure 7
- supplemental material - Supplemental Figure 8
- supplemental material - Supplemental Movie 1
- supplemental material - Supplemental Movie 2
- supplemental material - Supplemental Movie 3
