The Me31B DEAD-Box Helicase Localizes to Postsynaptic Foci and Regulates Expression of a CaMKII Reporter mRNA in Dendrites of Drosophila Olfactory Projection Neurons

mRNP granules at adult central synapses are postulated to regulate local mRNA translation and synapse plasticity. However, they are very poorly characterized in vivo. Here, in Drosophila olfactory synapses, we present early observations and characterization of candidate synaptic mRNP particles, one of which contains a widely conserved, DEAD-box helicase, Me31B. In Drosophila, Me31B is required for translational repression of maternal and miRNA-target mRNAs. A role in neuronal translational control is primarily suggested by Me31B's localization, in cultured primary neurons, to neuritic mRNP granules that contain: (i) various translational regulators; (ii) CaMKII mRNA; and (iii) several P-body markers including the mRNA hydrolases, Dcp1, and Pcm/Xrn-1. In adult neurons, Me31B localizes to P-body like cytoplasmic foci/particles in neuronal soma. In addition it is present to synaptic foci that may lack RNA degradative enzymes and localize predominantly to dendritic elements of olfactory sensory and projection neurons (PNs). MARCM clones of PNs mutant for Me31B show loss of both Me31B and Dcp1-positive dendritic puncta, suggesting potential interactions between these granule types. In PNs, expression of validated hairpin-RNAi constructs against Me31B causes visible knockdown of endogenous protein, as assessed by the brightness and number of Me31B puncta. Knockdown of Me31B also causes a substantial elevation in observed levels of a translational reporter of CaMKII, a postsynaptic protein whose mRNA has been shown to be localized to PN dendrites and to be translationally regulated, at least in part through the miRNA pathway. Thus, neuronal Me31B is present in dendritic particles in vivo and is required for repression of a translationally regulated synaptic mRNA.


INTRODUCTION
The formation of long-term memory (LTM) and the underlying persistent synaptic changes require protein synthesis at stimulated synapses (Steward and Schuman, 2001;Martin and Zukin, 2006;Sutton and Schuman, 2006;Costa-Mattioli et al., 2009;Richter and Klann, 2009). For this local translation, mRNAs have to be transported in a repressed state from soma to synapse, and then stored at sites where translational activation must occur. Several studies have implicated a role for ribonucleoprotein (RNP) particles in this process (Martin and Zukin, 2006;Sossin and DesGroseillers, 2006;Bramham and Wells, 2007;Banerjee et al., 2009;Mikl et al., 2010). RNP particles, which contain proteins involved in mRNA translation, transport, repression, and decay, can form large complexes that have been identified in neurons of different species by light microscopy (Krichevsky and Kosik, 2001; Barbee et al., 2006;Kiebler and Bassell, 2006;Cougot et al., 2008;Zeitelhofer et al., 2008;Miller et al., 2009;Tubing et al., 2010).
Staufen-and FMRP-containing RNPs in the neurites of cultured Drosophila neurons contain proteins found in P-bodies, somatic RNPs that are sites of mRNA degradation and translational control The Me31B DEAD-box helicase localizes to postsynaptic foci and regulates expression of a CaMKII reporter mRNA in dendrites of Drosophila olfactory projection neurons for P-body formation (Andrei et al., 2005;Coller and Parker, 2005). In contrast to these findings, which point to function as a negative regulator of mRNAs, observations in the C. elegans germline and in P. falciparum, indicates that the orthologs CGH1/DOZI proteins may have additional roles in stabilizing subsets of mRNAs and protecting them from degradation (Mair et al., 2006;Boag et al., 2008).
The Drosophila homolog Me31B is associated with maternal sponge bodies where it appears to repress translation of many granule-associated mRNAs (Nakamura et al., 2001). Me31B is associated with dFMR1/FMRP in neuritic mRNPs, regulates dendritic morphogenesis in sensory neurons, and, like its human homolog RCK, is required for optimal miRNA function (Barbee et al., 2006;Chu and Rana, 2006). Together, these observations point to a role for Me31B in the repression of dendritically localized mRNAs, their aggregation into transport mRNPs, and in control of locally translated synaptic mRNAs such as CaMKII, which regulate synaptic plasticity (Mayford et al., 1996;Aakalu et al., 2001;Ashraf et al., 2006).
However, many aspects of the above model have not been experimentally tested. It remains to be shown that mRNP aggregates exist in differentiated adult synapses in vivo. Therefore the composition and characteristics of such mRNP particles is unclear. In addition how Me31B in particular, and neuronal mRNP components in general, regulate target dendritic mRNAs also remains largely unknown in vivo.
To address these outstanding questions, we studied the neuronal expression and localization of Me31B in the adult Drosophila brain and the effect of Me31B mutations on the expression of synaptic CaMKII reporter protein (Mayford et al., 1996;Aakalu et al., 2001;Ashraf et al., 2006). We chose to analyze neurons and synapses in the olfactory circuit, which is one of the best-understood and genetically accessible neuronal networks in the adult brain (Davis, 2005;Vosshall and Stocker, 2007;Fiala, 2008). Not only is it well characterized in terms of its connectivity and function, but also the plasticity of synapses on projection neurons (PNs), as well as local translation of CaMKII mRNA in PNs, has been associated with olfactory memory (Ashraf et al., 2006;Keene and Waddell, 2007;Fiala, 2008).
Our data show: (i) that Me31B is expressed widely in the nervous system; (ii) that it is present on a postsynaptic particle in vivo; and (iii) that Me31B is required in PNs for the repression of CaMKII reporter mRNA. These observations represent an early step in the process of linking translational control proteins, synaptic mRNP dynamics and local mRNA translation with synaptic plasticity in a well-characterized behavioral circuit.

IMMUNOHISTOCHEMISTRy
Preparations of Drosophila adult brains were performed after R. Stocker (Stocker et al., 1990). Decapitated fly heads were fixed in 4% PFA in 1× PBS + 0.2% Triton for 3 h on ice, washed with 1× PBS + 0.2% Triton and brains were dissected in blocking solution (PBS + 0.2% Triton and 5% NGS). Brains were incubated with primary antibodies overnight on 4°C diluted in blocking solution, followed by incubation with secondary antibodies for 3 h at room temperature diluted in blocking solution.
For preparations of Drosophila antenna whole flies were fixed in 4% PFA in PBS + 3% Triton for 1 h on ice. Antenna were cut off and fixed for an additional 4 h on ice. The antenna were washed quickly with 1× PBS + 3% Triton for 3× 15 min, followed by a 2-day wash on 4°C. Incubation with primary antibodies in 1× PBS + 0.1% triton over night at 4°C and incubation with secondary antibody as well in 1× PBS + 0.1% triton over night at 4°C.
Preparations were mounted in Vectashield Mounting Medium (Vecta Labs) and imaged on a Zeiss LSM 510-meta confocal microscope.

IMAgE ACqUISITION
All images were taken on a Zeiss LSM 510 confocal microscope. To image mRNP particles in fixed brain tissue, we used a 63× objective (Zeiss Plan-Apochromat, 1.4 Oil Ph3) and the "digital zoom" software feature of the LSM 510 software (zoom factor between 3 and 4) that allows to scan a region of interest at a higher pixel resolution, constrained of course by the normal ∼300 nm resolution limit of light microscopy. Regions of particular interest were selected in Photoshop and presented in a way to allow a better evaluation by the reader.

MARCM
For the induction of MARCM clones Drosophila crosses of the respective genotype were raised at 25°C and the flies were transferred to a new vial every 4 h for timed egg collections. A heat-shock pulse at 37°C in a water bath for 1 h was given at optimal time points to induce single-cell clones in PNs projecting to the DL-1 glomerulus. The time points (between 0 and 60 h after larval hatching) were chosen after G. Jefferis (Jefferis Combining anatomical information with genetic markers for specific neuronal cell types, we directly confirmed Me31B expression in olfactory PNs (Figure 1D), local interneurons (LNs), and mushroom-body (MB) Kenyon cells ( Figure 1E). In addition to neurons, Me31B was also strongly expressed in glial cells, which sent processes into the AL ( Figure 1F). In control experiments, et al., 2001). Heat-shocked larvae were transferred back to 25°C, raised to adulthood and the adult flies were dissected 1-4 days after eclosion.

AUTOMATED SpOT COLOCALIzATION ANALySIS
We created "Spotnik" a MatLab plug-in which allowed us to automate the quantitative analysis of spot colocalization across red and green channels of a double-stained image. The details of the signalprocessing algorithms used have been described elsewhere (Pan et al., 2010). However, the main steps and logic were as follows.

spot region extraction
Given that spots can be seen in regions of the image with highly variable levels of local background labeling, a direct image thresholding tool does not allow spots to be identified. Thus, we created a spot extraction tool that sharpened and enhanced details of the image, using a Wiener filter coupled with a gamma operation, and then modified the contrast of the image such that local features have the same contrast independent of their local background. The image was now thresholded with a user-defined threshold, to create a mask that included regions well-contrasted from the background. This mask was then used to select regions of pixels from the original sharpened image for subsequent processing.

spot detection and analysis
Individual regions defined by the mask are first normalized to remove the background (by fitting a local plane to the background). A subsequent modeling step identified Gaussian parameters for each spot in each identified region using a Split-Merge Estimation-Maximization technique (Ueda et al., 2000) and synthesized Gaussians at their modeled positions. The center of these spots and their variances (widths) are then recorded for analysis. At each step, we ensured that manually detected spots were not lost and that computationally detected spots were not artifactual.

Colocalization analysis
Several different types of colocalization analysis are possible. However, for each spot imaged in one spectral channel, a simple MatLab tool allowed us to compute the distance of the closest spot in the other channel. Plots were then created in which the percent of spots that have corresponding closest spots closer than a given distance was plotted against distance.

Me31B IS ExpRESSED IN THE ADULT BRAIN
The expression of neuronal Me31B/DDX6/RCK/Dhh1/Cgh1 and the composition of associated neuronal mRNPs has been previously examined in dendrites of primary cultured neurons (Barbee et al., 2006;Cougot et al., 2008;Zeitelhofer et al., 2008;Miller et al., 2009). In order to extend these studies to an in vivo system, we used confocal microscopy and immunohistochemistry to examine Me31B expression in adult Drosophila brains, focusing primarily on the well-studied olfactory system ( Figure 1A). Me31B is ubiquitously expressed throughout the brain ( Figure 1B). Closer examination of Me31B expression in the antennal lobes (AL) area showed punctate expression in cell bodies and in the neuropil (synaptic) area of the AL ( Figure 1C).

FIguRe 1 | Me31B is ubiquitously expressed in the adult Drosophila brain. (A)
Cartoon schematic illustrating the main components of the olfactory system in the adult Drosophila brain (the inset shows a real stained brain; co-immunostaining for the presynaptic marker Nc82 (green) and Me31B (magenta)). Sensory information from the antenna is sent to the brain via olfactory sensory neurons (OSNs) which synapse in the antennal lobes (AL) to dendrites of projection neurons (PNs). The axonal terminals of OSNs and dendrites of PNs form dense synaptic areas called glomeruli. PN axons project into higher brain regions and form synapses in the mushroom-body (MB) calyx and the lateral horn. Olfactory information is believed to be stored in the MB. The sensory information can be regulated in addition by local interneurons (LNs), which form synapses with PNs and OSNs. (B) Projection of confocal images of an adult Drosophila brain (showing the region of the brain indicated in the inset above) stained with an Me31B antibody, showing apparently uniform Me31B expression in neuronal cell bodies throughout the brain. (C) A single confocal slice at a higher magnification shows the punctate expression of Me31B in cell bodies and as well as fainter foci in the synaptic neuropil (AL); inset shows an example of Me31B expression in the neuropil in higher magnification (gain adjusted for better visualization of foci in the neuropil). neuronal granule/transport mRNP marker Staufen, which may also be a component of P-bodies (Anderson and Kedersha, 2006;Eulalio et al., 2007a). In neuronal cell bodies, double-immunostained to visualize Me31B and P-body markers, we observed that a very large fraction of the Me31B-positive, cytoplasmic foci also contained each of the three proteins (Figure 2). This indicates that the Me31B foci in neuronal soma are similar to P-bodies and to mRNP granules in neurites observed in primary cell culture (Barbee et al., 2006). In contrast to particles in the soma, Me31B foci in the synaptic neuropil areas appeared to be distinct and to only rarely contain the P-body markers tested (Figure 3). Thus, although Dcp1, Pcm, or Stau-labeled foci were present in the neuropil, these showed relatively weak colocalization with Me31B.
In order to rigorously assess levels of colocalization, we developed an algorithm, tentatively named Spotnik that allowed us to efficiently identify foci in one spectral channel and to measure the distance to the closest foci in the other spectral channel. Two key features of this program were: (a) local field thresholding, which we confirmed that the anti-Me31B antibodies did not label cell bodies or synapses of me31B null mutant neurons (see Figure 6; Figure S1 in Supplementary Material). Thus, Me31B is present in both somatic and synaptic particles.
We further attempted to examine the composition of these particles and their relationship to P-bodies, cytoplasmic mRNP aggregates that mediate specific forms of translational repression and mRNA decay (Eulalio et al., 2007a;Parker and Sheth, 2007).

SOMATIC AND SyNApTIC Me31B fOCI DIffER IN THEIR COMpOSITION
In cultured Drosophila neurons, Me31B-positive mRNPs in neurites are related to P-bodies based on the high frequency at which they contain various P-body markers, including the RNA decapping enzymes Dcp1 and Pcm (the Drosophila homologue of the 5′-3′ RNA exonulease Xrn-1) (Barbee et al., 2006). We asked how these neuritic mRNPs compared with Me31B containing particles in vivo, by examining Me31B-positive foci in the adult Drosophila brain for presence of the P-body markers Dcp1 and Pcm as well as the  (Boag et al., 2008;Gallo et al., 2008). Alternative interpretations of these data are considered in the Section "Discussion".

Me31B foci are predoMinantly dendritic in oSns and pns
The observations above suggest an obvious hypothesis in which synaptic Me31B foci represent sites where mRNAs are held repressed until released for stimulation-induced, local translation. To ask whether Me31B particles were enriched in either the presynaptic terminal or dendrites, we performed a series of experiments, which examined the localization of Me31B foci relative to synaptic element or cell-type specific markers. In some experiments, we used the nc82 antibody to label Bruchpilot (Brp), a protein specific to presynaptic active zones. In others, we used cell-type specific expression of either a synaptotagmin-GFP (Syt-GFP) fusion protein to label presynaptic terminals, or a membrane associated myristoylated-GFP under control of the 3′UTR of Drosophila CaMKII (myr-GFP CaMKII3′UTR ) mRNA, shown to localize to dendrites (Ashraf allowed faint spots to be selected as efficiently as bright ones; and (b) Gaussian modeling of spots, which importantly allowed foci to be distinguished from shapeless or other local background signals. As can be seen from the image processing steps shown in Figure 4, the Gaussian modeling step also allowed two or three adjacent and overlapping foci to be resolved (see Materials and Methods; Pan et al., 2010). The distance between the centers of Gaussians (foci) in one channel to the closest Gaussian peak in the other were measured and the percentage of foci within a distance (x) of a foci in the other channel was plotted (Figures 4B-D).
The rigorous colocalization analysis enabled by Spotnik confirmed that particles labeled with Me31B colocalized much more efficiently with Dcp1, Pcm, and Staufen immunostained foci in cell bodies than it did at synapses. For example, based on using a 200-nm cut-off for perfect colocalization, Me31B showed 58% colocalization with Dcp1 in soma, but only 7% at the synapse. Thus, the data indicate that Me31B is present in a class of synaptic particle that lacks RNA degradative enzymes, reminiscent of a class of Confocal images of olfactory glomeruli in the Drosophila antennal lobe (AL) double-stained for Me31B (left panels or magenta) and P-body markers (middle panels or green). In contrast to Me31B-containing foci in the soma (Figure 2), synaptic Me31B particles show only weak colocalization with P-body proteins. Thus, in at least two of the major classes of AL neurons, Me31B foci are predominantly localized to dendrites.

Me31B REgULATES Dcp1 pOSITIvE pARTICLES IN pN DENDRITES
In non-neuronal cells, Me31B and its non-fly orthologs are required for the normal assembly of P-body like particles which contain not only Me31B, but also RNA hydrolases such as Dcp1 and Pcm (Barbee et al., 2006;Hillebrand et al., 2007). The observation that most dendritic Me31B-positive particles are not marked by anti-Dcp1 pointed to the possibility that dendritic Dcp1 particles may assemble in an Me31B independent et al., 2006). Figures 5A-C show that the latter reporter, when expressed in PNs, marks PN dendrites that show only minimal overlap with presynaptic elements in AL labeled with nc82.
In PNs, synaptic Me31B foci are predominantly found in postsynaptic dendrites (Figures 5D-F). Thus, in AL of animals expressing the myr-GFP CaMKII3′UTR reporter in PNs, Me31B foci showed considerable overlap with reporter GFP (Figure 5E). In contrast, Me31B foci were almost completely excluded from presynaptic endings of PNs visualized in the MB calyx (Figure 5F; 0.22 Me31B foci/μm 2 in PN dendrites in the AL versus 0.026 foci/μm 2 in PN terminals in the calyx). Me31B foci observed within AL glomeruli, but outside the areas labeled by the myr-GFP CaMKII3′UTR reporter in Figure 5E, are probably contained in processes that other cell types (e.g., LNs or glia) send to the AL.
In additional control experiments, we used the MARCM technique to analyze Me31B localization in single PNs as well as the specificity of Me31B staining in PN dendrites (Lee and Luo, 2001). The MARCM technique allows one to visualize individual, homozygous-mutant cells in otherwise heterozygous animals. We used the Gal4 lines NP3529, which among other tissues marks PNs specifically projecting into the glomerulus DL-1, and GH146, which marks about 60% of all PNs. Dendrites of individually imaged

FIguRe 4 | Computational analysis of Me31B colocalization with P-body proteins in somatic and synaptic foci. (A) Stages of image processing during automated detection of immunostained puncta (see Materials and Methods). The original image (i) is transformed into a mesh-plot (ii). The local background (iii) is subtracted from this image to yield image (iv). Each object in this backgroundsubtracted image is fit to Gaussian parameters using an SMEM (Split-Merge Estimation-Maximization) technique and the result (v) is used as a mask to analyze underlying spots in the original image (vi). (B-D)
Quantified colocalization of Dcp1, Pcm, and Staufen with Me31B. The graphs plot cumulative fraction of Dcp1, Pcm, or Stau positive particles found within a specific distance from the closest Me31B containing particle (fraction on the "y" axis is plotted versus distance on the "x"). Blue curves correspond to somatic puncta and red curves to synaptic ones (for each curve 20 images and about 5000 spots-per-channel, were analyzed). Distances are as measured by Spotnik from the computationally determined centroids for each spot, as described by the Gaussian modeling technique. However the actual position of these centroids are naturally limited by the resolution of light microscopy. Thus, spots separated by less than about 0.30 μ should be assumed to colocalize exactly. We examined Me31B and Dcp1 in single-cell clones of wildtype or me31b D2 /me31b D2 PNs generated using the MARCM technique. CD8-GFP marked, wild-type MARCM clones showed both somatic and dendritic Me31B and Dcp1 particles within areas marked by CD8-GFP (Figures 6A,E and not shown). And as expected, me31b D2 /me31b D2 mutant cells lacked Me31b-positive fashion. To address this specific question, as well as the more general issue of whether different classes of synaptic mRNPs interact and/or share common mechanisms for biogenesis, we examined the effect of loss of Me31B protein, on the brightness and number of Me31B or Dcp1 positive particles in olfactory PNs in vivo (Figure 6). (H) Me31B foci (magenta) are present in cell bodies of OSNs (green) and in OSN dendrites within the sensillum (arrowheads). Or83b-Gal4 driven GFP is used to reveal OSN soma and dendrites (green); and Me31B (magenta) is visualized by immunocytochemistry. (I) Presynaptic areas of OSNs (green) in the antennal lobes are marked by Or83b-Gal4 driven expression of synaptotagmin-GFP (Syt-GFP). Me31B foci (magenta) are often adjacent to, but rarely, if ever, within OSN presynaptic endings. This is consistent with predominantly somatodendritic localization of Me31B particles.

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November 2010 | Volume 4 | Article 121 | 8 Hillebrand et al. mRNPs in vivo in the Drosophila brains behavioral experience (Ashraf et al., 2006). Knockdown of Me31B in PNs caused a substantial increase in levels of CaMKII reporter protein in the AL (Figures 7D,E). Thus, Me31B is required in PNs for repression of a synaptically targeted, miRNA regulated mRNA. Me31B repression influences both dendritic and somatic levels of the CaMKII reporter protein; indeed, knockdown of Me31B is associated with roughly two-fold increase in levels of the reporter in PN cell bodies (not shown). This might be expected if Me31B is involved in the repression of CaMKII transcript in the soma in preparation for its dendritic targeting.

DISCUSSION
Experimental observations presented here are of interest because they: (a) provide new information on neuronal mRNPs in vivo; (b) reveal a novel function for Me31B in the regulation of dendritic mRNA expression; and (c) together highlight important aspects of mRNA regulation at synapses.

pROpERTIES AND fUNCTIONAL IMpLICATIONS Of SyNApTIC Me31B gRANULES OBSERvED in vivo
The Me31B/Dhh1p/DDX6/CGH-1 class of DEAD box helicases is associated with many different kinds of mRNP aggregates, including maternal RNA storage granules, P-bodies, stress granules, as well as various granule subtypes observed during foci in soma and dendrites (Figures 6B,C). In addition, these cells showed a striking reduction of Dcp1 foci (Figures 6D,F), which is quantified in Figure 6G. Thus, although synaptic Dcp1 foci appear to be largely free of Me31B (or to have particularly low levels of the protein), the integrity of these foci is dependent on Me31B.

Me31B REpRESSES A CaMkII REpORTER mRNA IN pN DENDRITES
The presence of Me31B in postsynaptic particles suggested that the protein might repress synaptic mRNA translation, similar to its role in oskar and bicD regulation. We therefore asked whether loss of Me31B would result in significant upregulation of CaMKII translation in dendrites, much as its loss causes precocious translation of Oskar and BicD in oocytes (Nakamura et al., 2001;Ashraf et al., 2006). We knocked down Me31B in PNs by coexpressing two doublestranded RNAi constructs that have been previously used to knockdown Me31B in non-neuronal cells (Barbee et al., 2006;Hillebrand et al., 2007) and analyzed the effects of this knockdown on expression of a translational reporter for the CaMKII mRNA (Ashraf et al., 2006) (Figures 7D,E). In the CaMKII reporter, GFP coding sequences are placed under control of the 3′UTR for CaMKII. When transcribed in PNs, the reporter mRNA is transported to PN dendrites where it is repressed by the miRNA pathway and activated by C. elegans germline development (Nakamura et al., 2001;Cougot et al., 2004;Andrei et al., 2005;Coller and Parker, 2005;Kedersha et al., 2005;Eulalio et al., 2007b;Boag et al., 2008;Jud et al., 2008;Noble et al., 2008). In addition it is required for the assembly of P-bodies in yeast, Drosophila and mammalian cells (Andrei et al., 2005;Coller and Parker, 2005;Eulalio et al., 2007b;Hillebrand et al., 2007) and as well for the formation of stress granules in mammals (Wilczynska et al., 2005). For these reasons, the punctate distribution of Me31B in postsynaptic dendrites is likely to indicate its presence in a specific type of synaptic mRNP particle. However, unlike Me31B-positive particles described in neurites of cultured Drosophila neurons, synaptic Me31B foci do not appear to contain the RNA hydrolases Dcp1 and Pcm/Xrn-1. Thus, they may be a distinct class of particle, which localize preferentially to postsynaptic dendrites. These represent early images of candidate mRNA storage particles at synapses in vivo (Christie et al., 2009). A paucity of antibodies and the challenging nature of such high-resolution immunocytochemistry in whole brain tissue has so far made it difficult to more completely characterize other components of synaptic Me31B particles as well as to establish whether Dcp1, Pcm, and Stau coexist on the same or different particle in the adult brain. Indeed even our conclusion that Me31B particles constitute a separate class must be qualified by the possibility that the visualization of two apparently distinct particle types arises from an artifact of incomplete antibody penetration into the neuropil. Our observations in neurons indicate a function for Me31B in repressing translation of a miRNA regulated, dendritically localized reporter mRNA in vivo. This is consistent with two related lines of data. First, it is consistent with the known function for Me31B in repression of miRNA-target genes in Drosophila wing imaginal cells as well as for its human homolog RCK in mammalian cultured cells (Barbee et al., 2006;Chu and Rana, 2006). Second, the correlation we observe between loss of synaptic Dcp1 puncta and upregulation of the CaMKII reporter, is consistent with observations in hippocampal cultured cells, where observed disassembly of "dendritic P-bodies" induced by synaptic stimulation has been proposed to underlie the temporally coincident translation of localized mRNAs (Zeitelhofer et al., 2008).
Thus, we suggest a simple model in which neuronal Me31B, as well as its homologs in other metazoa, mediates the formation of synaptic mRNP particles that contain locally repressed mRNAs. And that synaptic stimulation-induced disassembly of these particles is one aspect of the mechanism of local translational control.

fUTURE DIRECTIONS
One key goal of future studies will be to understand the composition and dynamics of dendritic mRNPs in vivo. This will be aided by genetic techniques to replace endogenous translational control molecules with genetically encoded, fluorescently tagged variants that retain functional and localization patterns of the endogenous proteins. When coupled with procedures to induce local protein synthesis in dendrites, such reagents will allow analysis of functionally relevant particle dynamics in vivo. In addition, by eliminating the need for antibodies whose use may be associated with artifacts of inclusion and exclusion, such reagents may provide more direct insight into the real nature of synaptic mRNPs in vivo.
A second goal is to understand the mechanism by which Me31B regulates the expression of CaMKII reporter levels in vivo. Although Me31B has been shown to be required for the miRNA pathway (Barbee et al., 2006;Chu and Rana, 2006) it is also required for other forms of translational repression, for example in S. cerevisiae that does not have miRNAs. Similarly, although the reporter used here is miRNA regulated, the same UTR also has binding sites for translational regulators that may operate independently of miRNAs (Ashraf et al., 2006). Thus, important and linked goals of future studies are to understand mechanisms by which the CaMKII UTR is regulated in dendrites and how Me31B engages with these mechanisms of neuronal translational control.
It is possible that synaptic Me31B particles could be analogous to recently described granules in the C. elegans germline, which contain translationally controlled mRNAs and CGH-1/Me31B but exclude decapping enzymes and the P-body protein PATR1/PAT1 (Boag et al., 2008;Gallo et al., 2008). Immunoprecipitation and further colocalization studies suggest that these granules can also contain PAB-1, ATX-2, or TIA-1, markers of stress granules, which in other systems, contain translation initiation factors together with mRNAs stalled in translational initiation (Kedersha et al., 2005;Gallo et al., 2008). Thus, it is conceivable that Me31B/CGH-1containing storage particles contain mRNAs stored in a stressgranule like state, in which the resident mRNAs are available for rapid activation (Rajyaguru and Parker, 2008).
The potential separation of storage and degradative particles leads to an attractive model in which individual mRNAs may transition from being available for translational activation in a storage granule, to being targeted for degradation in a P-body like particle. This is supported as well by observations in dendrites of cultured mammalian neurons where a distinct class of RNPs contain the degradative enzyme Xrn1, which is excluded from RNPs supposedly involved in storage (Cougot et al., 2008).
At synapses, a transition between storage and degradation particles may occur by three, non-exclusive, candidate mechanisms: (a) by the remodeling of a storage mRNP to a degradative one through protein exchange; (b) by the initial exit of mRNA from the storage RNP to a translating pool, followed by its subsequent targeting to a degradative particle; or (c) the fusion of the two particles. Recent studies in Drosophila provide a possible mechanism by which a change of proteins in RNP complexes could alter its function. Two related proteins of the Lsm-family, Enhancer of Decapping 3 (EDC3), which is implicated to play a role in mRNA decay, and Trailer Hitch (Tral), which supposedly is involved in mRNA repression, interact at the same domain with the Me31B protein. This suggests that the function of Me31B complexes might be determined by the interaction with specific binding partners (Tritschler et al., 2008(Tritschler et al., , 2009. Some support for the second model is provided by the observation that the synaptically localized Arc mRNA is targeted for degradation after its translation is induced by synaptic activity (Bramham et al., 2008) and also by the observation that RCK-positive particles in dendrites of cultured hippocampal neurons are transiently disassembled following BDNF stimulation (Zeitelhofer et al., 2008). Further studies are required to understand how, when, and even whether these transitions of mRNA state occur in synapses and other biological contexts.

fUNCTIONS Of Me31B IN NEURONAL TRANSLATIONAL REpRESSION
Together with many analogous studies in yeast and mammalian cells, previous observations in Drosophila that Me31B is a repressor of maternal mRNA translation, a component of a repression pathway mediated by the bantam microRNA, and a repressor of growth of terminal dendrites, has led to a strong model that Me31B is a translational repressor protein (Nakamura et al., 2001;Coller et al., 2002;Barbee et al., 2006;Chu and Rana, 2006). In contrast, recent studies in C. elegans and P. falciparum have shown that Me31B orthologs, CGH-1 and DOZI, associate with specific mRNAs and protects them from degradation (Mair et al., 2006;Boag et al., 2008).

FIguRe S2 | (A)
To test the significance of the quantitative data for Dcp1, Pcm, and Staufen coexpression with Me31B, presented in Figure 4, we divided the raw data into two classes, spot distance below 400 nm and spot distance between 400 nm and 1 μm. The graphs in (A) show the percentage of Dcp1, Pcm, and Staufen foci which are found in a distance to the nearest Me31B foci below or above 400 nm. A Student's t-test was used to analyze the statistical significance of differences between somatic and synaptic coexpression. For all three proteins tested, the difference between somatic and synaptic coexpression is highly significant (P-value <0.0001, n = 20). (B) To analyze the specificity of "Spotnik" , we performed an antibody staining against Me31B using a primary rabbit antibody and two secondary anti rabbit IgG antibodies (Alexa anti-rabbit 488 and 555). The quantification of coexpression between 488 and 555 in the soma and the neuropil showed a high colocalization (69% of foci below 400 nm apart in the soma and neuropil) and we did not find any significant differences between somatic and synaptic coexpression.