PMC

Transport of CPEB-GFP particles. Confocal time-lapse images of CPEB-GFP particles in transfected hippocampal neurons depicting a retrograde movement. V_max and V_avg are the maximal and average velocities, respectively, of indicated particle movement. Bar, 5 μm.

CPEB-GFP forms particles and colocalizes with RNA and maskin. (A) Colocalization of CPEB-GFP and CPE-containing RNA in B104 cells. CPEB-GFP plasmid DNA and Alexa-546-labeled 3CPE-containing or -lacking polylinker RNA were coinjected into B104 cells and imaged by dual-channel confocal microscopy. Particles containing either CPEB-GFP (green) or Alexa-546-labeled RNA (red) are denoted by open arrows; particles containing both CPEB-GFP and RNA (yellow) are denoted by solid arrows. (B) Hippocampal neurons were cotransfected with CPEB-CFP (green) and YFP-maskin (red) and imaged by confocal microscopy. The arrows in the magnified regions denote particles that contain both CPEB-CFP and YFP-maskin proteins. (C) Colocalization of CPEB-CFP and YFP-maskin in 620 RNA particles from dendrites of eight double-transfected cells was quantified by single-particle ratiometric analysis. The two broken lines define the region in which CPEB and maskin in particles are present within 1:2 and 2:1 intensity ratios.

Deficient transport of CPE-containing RNA in neurons from CPEB knockout mice. (A) Wild-type (CPEB+/+) and knockout (CPEB−/−) hippocampal neurons were infected with Sindbis virus expressing GFP-3CPEs. The neurons were subjected to in situ hybridization for GFP RNA, and also costained with MAP2 antibody. (B) The quantification of the in situ hybridization signal for GFP was performed as in Figure B. (C) The mean intensity of each 10-μm dendritic segment was calculated from 20 CPEB+/+ and 20 CPEB−/− neurons expressing GFP-3CPE and presented in histogram form; error bars show the S.E.M. The difference in hybridization signal between the two RNAs is significant (p < 0.05, Student's t test). Bars, 10 μm.

The CPE is sufficient for dendritic RNA transport. (A) Hippocampal neurons were infected with recombinant Sindbis virus as noted above, but in this instance, the GFP 3′-UTRs consisted of a polylinker sequence (GFP), or a polylinker containing three CPEs. The neurons were subjected to in situ hybridization for GFP RNA, and also costained with MAP2 antibody. (B) The quantification of the in situ hybridization signal for GFP was performed as in Figure B. (C) The mean intensity of each 10-μm dendritic segment was calculated from neurons expressing GFP-3CPE (11 dendrites) and GFP (10 dendrites) and presented in histogram form; error bars show S.E.M. The difference in hybridization signal between the two RNAs is significant (Student's t test). Bars, 10 μm.

A CPEB mutant is defective for kinesin and dynein interaction. (A) Salient features of CPEB showing the LDSR aurora phosphorylation site, a PEST instability region, two RNA recognition motifs (RRMs) and two zinc fingers (Zif). The structures of various CPEB mutant proteins are also shown, as well as whether they form granules, are transported to neurites, or are nuclear or cytoplasmic. (B) Localization of wild-type and mutant CPEB-GFP fusion proteins in transfected B104 cells. (C) Western blot of wild-type and mutant CPEB-GFP proteins in transfected B104 cells. (D) Gel shift using extracts from B104 cells transfected with wild-type or mutant CPEB-GFP fusion proteins with CPE-lacking (left) or CPE-containing (right) RNA. The asterisks denote specific shifts attributed to CPEB proteins. The arrow denotes a nonspecific band caused by an unknown protein from B104 cells. (E) Coimmunoprecipitation of wild-type or RBD mutant CPEB-GFP fusion proteins with motor proteins. Cells infected with DNA encoding the heterologous proteins noted above were immunoprecipitated, washed, and eluted with SDS loading buffer. The eluates were probed for CPEB-GFP and RBD-GFP (left) as well as kinesin heavy chain and dynein intermediate chain.

Facilitation of mRNA transport by the CPE. (A) Hippocampal neurons were infected with Sindbis virus carrying GFP fused to a 170-bp 3′-UTR of wild-type αCaMKII that contains two CPEs, or a mutated αCaMKII 3′-UTR in which the CPEs have been mutated. Seven hours after infection, the neurons were fixed for in situ hybridization with GFP sequences, followed by the MAP2 immunostaining. The Northern blot shows comparable expression of the two constructs in infected neurons. (B) Quantification of dendritic GFP mRNA as assessed by in situ hybridization. The average GFP RNA signal (wild-type or mutant αCaMKII 3′-UTR) in each segment of one major dendrite from each neuron was measured (NIH-Image) and plotted against the distance of the signal from the soma. (C) The mean intensity of each 10-μm dendritic segment was calculated from neurons expressing GFP-αCaMKIIwt (31 dendrites) or GFP-αCaMKIImut (27 dendrites) and presented in histogram form; error bars show S.E.M. The difference in the amount of dendritic GFP hybridization signal between the two RNAs is significant (p < 0.05, Student's t test). Bars, 10 μm.

CPEB-GFP particles contain dynein and kinesin and are transported in a microtubule-dependent manner. (A) CPEB-GFP transfected hippocampal neurons were treated with latrunculin A, which disrupts microfilaments, or vincristine, which disrupts microtubules, and examined for GFP fluorescence (green) as well as tubulin immunofluorescence (red). Only vincristine disrupted most microtubules by leaving a patchwork of these structures, whereas neither treatment affected dendrite integrity (merged DIC image). Vincristine, but not latrunculin A, also inhibited GFP-CPEB transport. (B) CPEB-GFP-expressing neurons were immunostained with kinesin or dynein antibody and imaged by confocal microscopy. The magnified images (insets) show particles containing CPEB-GFP colocalized with motor proteins (solid arrows). (C) The green and red fluorescence intensities representing CPEB-GFP and either kinesin or dynein were analyzed from 407 and 403 dendritic CPEB-GFP-containing particles, respectively. The intensity of kinesin or dynein in each particle was plotted against the intensity of CPEB-GFP in the same particle. The two broken lines define the region in which CPEB and the molecular motors in particles were detected in 1:2 and 2:1 intensity ratios. Bars, 10 μm. (D) Coimmunoprecipitation of kinesin and dynein with CPEB-myc13. Cytoplasmic extracts prepared from hippocampal neurons, either uninfected or infected with Sindbis virus-expressing myc-tagged CPEB, were immunoprecipitated with myc antibody in the absence or presence of RNase A. The antibody beads were washed, eluted with 2 M KCl, and probed on a Western blot for kinesin and dynein, as well as for CPEB. For reciprocal immunoprecipitation experiments, the extract from CPEB-myc13-infected neurons was incubated with monoclonal antibodies against cytoplasmic dynein intermediate chain or kinesin heavy chain; mouse IgG served as a negative control. The immunoblots were probed with antibodies against CPEB-myc13, dynein, and kinesin. The asterisk denotes the breakdown product of CPEB-myc13.

CPEB facilitates RNA transport to dendrites. (A) Infection of hippocampal neurons with Sindbis virus harboring RBD-GFP (RBD refers to the RNA binding region of CPEB) and CPEB-GFP, which were also immunostained with antibody directed against MAP2. Note that whereas CPEB-GFP was detectable in the soma and dendritic arbors, RBD-GFP was confined to the soma. Bars, 10 μm. (B) The extracts from neurons infected with recombinant Sindbis virus were immunoblotted for GFP and CPEB-GFP fusion proteins. The bottom portion of the panel shows the degree of synaptoneurosome enrichment following infection with recombinant virus. Using a densitometric scan of a Western blot probed for αCaMKII, the degree of enrichment was comparable for each preparation. (C) Key features of neurofilament-M (NF-M), Arc, and dendrin mRNAs, which lack obvious CPEs, and MAP2, αCaMKII, and IP3 receptor mRNAs, which contain multiple putative CPEs. Synaptoneurosomes were prepared from GFP-, CPEB-GFP-, and RBD-GFP-containing Sindbis virus-infected cells, and the amount of the RNAs noted above was quantified by real-time PCR. To take into account small variations of the amount of cDNA in each preparation, the amount of RNA in synaptoneurosomes following GFP-expressing virus infection was used as the normalization standard. Error bars indicate the S.E.M., and the asterisks denote significance (p < 0.05, Student's t test). (D) The gel at the bottom shows the final PCR amplification products. (E) In situ hybridization of MAP2 RNA. Hippocampal neurons were infected with virus containing GFP, CPEB-GFP, or RBD-GFP and processed for in situ hybridization for MAP2 RNA. The intensity of the hybridization signal is color-coded. On the same coverslip, the MAP2 RNA hybridization signals were compared between GFP-infected and uninfected neurons, CPEB-GFP-infected and uninfected neurons, and RBD-GFP-infected and uninfected neurons, quantified, and plotted as a ratio of the two (right). The differences between the MAP2 RNA signals in dendrites are significantly different between CPEB-GFP-infected and uninfected neurons, and between RBD-GFP-infected and uninfected neurons (p < 0.05, Student's t test). The GFP fluorescence in dendrites from infected neurons is also shown.