Dendritic Localization of the Translational Repressor Pumilio 2 and Its Contribution to Dendritic Stress Granules

Synaptic activity increases Pum2 protein levels in neurons

Synaptic activity is known to increase the synthesis of mRNA in neurons (Wu et al., 1998). Because Pum2 is known to act as a translational inhibitor in Drosophila (Parisi and Lin, 2000) and translational inhibitors are likely necessary for preventing the improper translation of mRNA during RNP transport (Dahm and Kiebler, 2005), we hypothesized that glutamate stimulation of cultured neurons would result in an increase in Pum2 protein. Neurons were incubated overnight in a mixture of synaptic inhibitors (1 μm TTX, 50 μm APV, and 100 μm CNQX). After this treatment, neurons were subjected to stimulation with 10 μm glutamate in the presence or absence of the inhibitor mixture. This stimulation with glutamate led to a significant increase in Pum2 protein in the cell body (Fig. 3C, left graph). The average fluorescence intensity, when normalized to the control condition consisting of antagonist only, was significantly increased by 91.0 ± 11.0% (p < 0.01). This finding could be attributed to either new Pum2 protein production or an increase in somatic Pum2 caused by additional protein being recruited from the distal dendrites. To clarify which mechanism was responsible, we repeated the experiments and analyzed Pum2 protein levels via Western blotting (Fig. 3C, right graph). When subjected to fluorescence intensity analysis and normalized to tubulin protein levels, it was evident that the increase in Pum2 fluorescence in the soma arises because of an overall increase in Pum2 protein levels. When treated for 2 min with 10 μm glutamate and then washed in HBSS for 10 min, Pum2 protein increased approximately twofold. After 20 min, Pum2 protein levels had increased threefold over control conditions.

Pum2 is found in ribonucleoparticles and is recruited into dendritic stress granules

To better characterize the molecular properties of Pum2, we fractionated brain cytoplasmic extracts on a 15–50% sucrose density gradient. Fractions were recovered and analyzed by Western blotting with anti-Pum2, anti-Staufen1, and anti-L7a antibodies. In the presence of either cycloheximide or puromycin (data not shown), a significant amount of Pum2 was found in fractions of lower density devoid of ribosomes. In contrast to Stau1, Pum2 did not cofractionate with the major peak of ribosomes (Fig. 4A). These data suggest that Pum2 does not associate with polyribosomes or ribosomal subunits and thus behaves like Smaug, another RNA-binding protein that acts as a translational inhibitor (Baez and Boccaccio, 2005).

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Figure 4.

Pum2 is a component of RNPs and forms SGs after disruption of translation. A, Fractionation of adult rat brain extract by 15–50% sedimentation sucrose gradient centrifugation. A Western blot was performed on fractions to detect Pum2, Staufen1, and the large ribosomal subunit L7a. B, Dendritic Pum2 puncta contain mRNA. Immunostaining for Pum2 (green) was combined with FISH using an oligo-dT probe to detect mRNA. Pum2-containing puncta showed strong colocalization with mRNA (red). Some Pum2 puncta did not contain mRNA (arrows), whereas all mRNA contained Pum2 (arrowheads). C, Puromycin treatment alters the cellular distribution of Pum2 particles. Hippocampal neurons were treated with 20 μg/ml puromycin for 4 h and immunostained for Pum2 (Pur). After drug treatment, Pum2 formed large aggregates in the cell body. Similar rearrangement occurs in dendrites (arrowheads). In contrast, no changes in Pum2 subcellular localization was observed in neurons treated with cycloheximide (Chx). UT, Untreated. Scale bar, 5 μm.

To determine whether the small Pum2 puncta observed in dendrites contain mRNA and can be considered RNPs, fluorescence in situ hybridization (FISH) using oligo-dT probes was performed in combination with Pum2 immunocytochemistry (Fig. 4B). In distal dendrites, all mRNA puncta contained Pum2; however, not all Pum2 puncta colocalized with mRNA. These data indicate the majority of Pum2 particles represent RNPs, a finding similar to other RNA binding proteins such as Staufen (Monshausen et al., 2001; Tang et al., 2001; Mallardo et al., 2003) and Barentsz (Btz) (Macchi et al., 2003).

The PuF family of proteins act as inhibitors of translation in many organisms (Parisi and Lin, 2000; Baines, 2005; de Moor et al., 2005). Because Pum2-associated polyadenylated mRNA might be silenced for translation or associated with translating ribosomes, we therefore investigated the effects of protein synthesis inhibitors that affect polysome stability on Pum2 distribution. We incubated hippocampal neurons in the presence of puromycin followed by immunostaining for Pum2 (Fig. 4C). Puromycin treatment caused a significant rearrangement of the endogenous Pum2 particles. In contrast to untreated neurons that showed small Pum2 puncta distributed homogeneously throughout the somatic cytoplasm, treatment of neurons with puromycin led to the formation of larger granules. Similar changes in Pum2 distribution were also observed in dendrites, in which Pum2 particles were clustered into larger aggregates along the dendritic shaft (Fig. 4C, dendrite). In contrast, treatment with cycloheximide (10 μg/ml) did not alter the Pum2 staining pattern (Fig. 4C). The large granules that formed in the presence of puromycin were reminiscent of SGs. It has been reported that various cellular stresses, including puromycin treatment, induce the accumulation of a number of RNA-binding proteins such as Staufen (Thomas et al., 2005), FMRP (Mazroui et al., 2002), and elements of the translational machinery including PABP and eIF4E into SGs (Kedersha et al., 2005).

To determine whether metabolic stress indeed recruited Pum2 into SGs, we treated hippocampal neurons with 500 μm arsenite for 60 min. We first tested the staining pattern of TIAR, an RNA-binding protein that is a well established marker for SGs (Kedersha et al., 1999), before and after treatment with arsenite (Fig. 5A). In the absence of arsenite, TIAR is primarily found in the nucleus with weak homogeneous staining detectable in both the cell body and dendrites. After arsenite treatment, discrete granules were observed in both compartments, suggesting that TIAR, primarily a nuclear protein, was recruited to distal dendrites (Fig. 5A, arrowheads). As shown in Figure 5A, these large granules also contain Pum2. Interestingly, Pum2/TIAR-positive granules were detected in dendrites (Fig. 5A, enlargements). To illustrate the degree of colocalization along the length of a representative dendrite, line-scan profiles of the fluorescence intensity of both fluorophores (the red and green channels) are shown in supplemental Figure 1 (available at www.jneurosci.org as supplemental material). To further clarify that these aggregates containing Pum2 were indeed SGs, we tested for other known components of these structures. After induction of SG formation, we performed immunostaining for Pum2 together with eIF4E or FMRP, two proteins previously shown to be present in SGs (Mazroui et al., 2002; Kimball et al., 2003; Kedersha et al., 2005; Kim et al., 2006). We found that, like TIAR, both proteins were present in Pum2-containing SGs in both the cell body and dendrites (Fig. 5B).

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Figure 5.

Metabolic stress recruits Pum2 into stress granules. A, Fifteen DIV hippocampal neurons were stained for TIAR (green) and Pum2 (red) before (top) or after (bottom) treatment with 500 μm arsenite for 1 h. In the absence of stress, TIAR is primarily nuclear with low levels of diffuse staining in the soma and dendrites. In the presence of stress, Pum2 colocalizes with TIAR in both the cell body (shown at a lower exposure in the top or bottom corners) and distal dendrites (see enlargements and arrowheads). B, eIF4E and FMRP also colocalize with Pum2 after arsenite treatment. This colocalization occurs in both the cell body (top) and in dendrites (bottom). Scale bar, 10 μm.

To determine whether Pum2-positive SGs also contained mRNA, we performed immunostaining combined with FISH using labeled oligo-dT probe in stressed and unstressed neurons (Fig. 6A). Pum2-containing SGs showed a high degree of colocalization with mRNA-containing granules (see also supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Together, these data demonstrate that Pum2 represents a novel component of SGs in both the cell body and dendrites. This is the first evidence that dendritic SGs exist, supporting the idea that a stress-related translational control mechanism is present in dendrites in which localized mRNA undergoes local protein synthesis in hippocampal neurons (Steward and Schuman, 2001).

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Figure 6.

A, Pum2 and mRNA are found in neuronal SGs. After metabolic stress, immunostaining for Pum2 (green) combined with FISH was performed. To detect messenger RNA, an oligo-dT probe was used. Pum2-containing SGs showed substantial colocalization with mRNA (red). Immunostaining with anti-TIAR antibody (green) combined with FISH to detect mRNA (red) served as a positive control (data not shown). Pum2-containing SGs are dynamic structures. Hippocampal neurons were cotreated with arsenite and either cycloheximide or puromycin and then immunostained for Pum2 (red) along with either TIAR or eIF4E (green). Cycloheximide (B) but not puromycin (C, D) dissolves Pum2-containing SGs in the presence of arsenite. Scale bar, 10 μm.

We then tested whether Pum2-containing SGs were static after formation or dynamic structures (Kedersha et al., 2000). Stress was induced with arsenite treatment in the presence of cycloheximide or puromycin (Fig. 6B,C). In contrast to puromycin, which causes premature chain termination releasing ribosomes from mRNA, cycloheximide stabilizes polysomes by inhibiting elongation. Treatment with arsenite in the presence of puromycin did not interfere with the recruitment of Pum2 into SGs (Fig. 6C). Similar results were obtained after staining with Pum2 and eIF4E (Fig. 6D). In contrast, cycloheximide prevented the formation of Pum2-positive SGs (Fig. 6B). Because cycloheximide, like emetine (Kedersha et al., 2000), traps mRNA in polysomes, it prevents the de novo formation of SGs in the presence of the stress stimuli. These data therefore suggest that, during stress, Pum2-containing SGs are in dynamic equilibrium with polysomes.

Pum2 is excluded from P-bodies

SGs are functionally distinct from P-bodies, which are cytoplasmic sites of mRNA storage and degradation (Sheth and Parker, 2003; Cougot et al., 2004, Liu et al., 2005). Neurons were transfected with a construct expressing enhanced green fluorescent protein (EGFP)-tagged Dcp1a, a known component of P-bodies (Lykke-Andersen, 2002), to determine whether Pum2 also associates with these structures. Few discrete Dcp1a–EGFP granules were found in dendrites of untreated neurons (Fig. 7A, −arsenite). These granules, in contrast to TIAR-containing SGs, did not colocalize with Pum2 either in the presence or absence of arsenite (Fig. 7, enlargement; see also supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Interestingly, during stress treatment, we observed a twofold increase in the number of Dcp1a–GFP granules in distal regions of the dendrites. In the absence of stress, there were 0.38 ± 0.032 Dcp1a–GFP puncta per 10 μm of dendrite length. In the presence of arsenite, the number of puncta increased to 0.78 ± 0.064 Dcp1a–GFP puncta per 10 μm of dendrite length (p < 0.001). However, these granules still did not colocalize with Pum2-containing SGs (Fig. 7A).

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Figure 7.

Pum2 is excluded from P-bodies. A, Fifteen DIV hippocampal neurons were transiently transfected with Dcp1a–GFP, a marker for P-bodies. The neurons were either left untreated (top) or treated with 0.5 mm arsenite for 1 h (bottom). Under both conditions, Pum2 is not found in P-bodies. After metabolic stress, the number of P-bodies in distal dendrites increased (arrowheads). B, Endogenous P-bodies as visualized with Lsm1 antibodies also do not contain overexpressed Pum2 tagged with EYFP. The enlargements show colocalization of Pum2 (green) and TIAR (blue), but not with Lsm1 (red). Scale bar, 10 μm.

To further illustrate that Pum2 was found in SGs but was excluded from P-bodies after arsenite treatment, we expressed GFP-tagged Pum2 in hippocampal neurons followed by immunostaining for both TIAR and Lsm1, a protein found in P-bodies (Ingelfinger et al., 2002). Stress induction led to the formation of large TIAR-positive aggregates of Pum2–EYFP along dendrites (Fig. 7C). In contrast, Lsm1 granules were adjacent to, but not colocalizing with, Pum2–EGFP (Fig. 7C). These data show that Pum2 is specifically recruited to SGs but not P-bodies in dendrites of hippocampal neurons.

Pum2 facilitates SG formation

To analyze the role of Pum2 in SG formation, full-length Pum2 (Pum2_FL) (for a scheme, see Fig. 8A) tagged with EYFP was expressed under the control of the cytomegalovirus promoter (Fig. 8B,C) in HeLa cells. It was found that even in the absence of stress stimuli, when fixed 18–24 h after transfection and stained for TIAR, 74.5 ± 1.2% of transfected cells form Pum2-containing SGs that are also positive for TIAR (Fig. 8C, FL). Eighteen to 24 h after transfection and after induction of metabolic stress with arsenite, Pum2–EYFP was recruited into SGs in >95% of transfected cells (data not shown). The addition of cycloheximide dispersed SGs induced by Pum2 overexpression (data not shown), again suggesting that these structures are dynamic. Similar observations were made in neurons in which granules formed by overexpression of Pum2_FL also led to the recruitment of other RNA-binding proteins such as Staufen1 and Btz (supplemental Fig. S2B, available at www.jneurosci.org as supplemental material). These proteins are involved in RNA transport and nonsense-mediated decay (Palacios et al., 2004; Kim et al., 2005). This further indicates that the granules induced after overexpression are comparable with SGs formed during the stress response. However, in contrast to this observation, staining for endogenous Pum2 after transfection with cytoplasmic polyadenylation element-binding protein (CPEB)–GFP and Maskin–cyan fluorescent protein (CFP) and treatment with arsenite demonstrated that these two RNP proteins were not recruited to the same granules as Pum2 (supplemental Fig. S3A, available at www.jneurosci.org as supplemental material). Together, this is the first indication that Btz is a component of SGs and also demonstrates that there may be multiple subtypes of SGs in neurons.

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Figure 8.

Overexpression of Pum2 is sufficient for SG induction. A, Scheme of the Pum2 constructs transfected in HeLa cells. B, HeLa cells were transiently transfected with full-length Pum2 constructs, or N- or C-terminal deletion mutants tagged with EYFP. The cells were fixed 20 h after transfection and immunostained for TIAR (red). The subcellular distribution of the fusion proteins was analyzed by fluorescence microscopy. Full-length Pum2 (FL; Bi) induces the formation of TIAR-positive SGs. Both the C-terminal (650–Stop; Bii) and N-terminal (1–651; Biii) region are also capable of inducing SG formation after overexpression. Scale bar, 10 μm. C, Quantification of three independent experiments demonstrates that the three constructs significantly induce SG formation after overexpression. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.0001. D, Time course of SG formation in neurons. Neurons expressing Pum2_FL–EYFP were exposed to arsenite (at time point 0) and imaged over 57 min. SG formation is first detected in the cell body and dendrites (arrowheads) ∼16 min after the addition of arsenite. The phase contrast image in the top left corner shows the neurons at the beginning of the time-lapse video before the addition of arsenite. The asterisks indicate SGs in proximal dendrites. Scale bars: bottom left, 10 μm; enlargements, 5 μm. Videos are available on request.

To determine which region of the Pum2 protein is responsible for the induction of SGs, we generated different fluorescence-tagged fusion constructs: the N-terminal region (Pum2_1–651; amino acids 1–651) and the C-terminal portion (Pum2_650-Stop) that contains the RNA-binding Pumilio homology domain (PHD) (Fig. 8A). Because the C-terminal PHD is responsible for RNA binding, we expected to observe SG formation during its overexpression, and the transfection of Pum2_650-Stop did induced SG formation in 73 ± 1.2% of transfected cells (Fig. 8Bii). A significant nuclear accumulation was also observed. After induction of stress, Pum2_650-Stop was found in discrete granules positive for TIAR in almost 100% of transfected cells (data not shown). The overexpression of Pum2_1–651 also induced SG formation (Fig. 8Biii). The number and the morphology of Pum_1–651 granules were, however, different when compared with endogenous SGs or SGs induced by overexpression of Pum2_FL and Pum2_650-Stop. They were fewer in number and consistently smaller in size. The majority of the fluorescence signal displays a homogeneous cytoplasmic distribution. Similar results were observed when Pum2_FL, Pum2_1–651, and the Pum2_650-Stop constructs were overexpressed in neurons, suggesting that the ability of Pum2 to induce SG formation is not specific to a certain cell type. A quantification of three independent experiments in HeLa cells is shown in Figure 8C.

We then set out to determine the time course of SG formation after initiation of the stress response. HeLa cells and neurons were imaged 6–8 h after transfection of Pum2_FL, before SGs formed, and 500 μm arsenite was added to neurons displaying transfected and untransfected cells (Fig. 8D). Images were acquired every 30 s for 57 min. Discrete aggregates of the fluorescently tagged Pum2 protein were evident in the cell soma and dendrites at 16 min. This time course of neuronal SG formation is comparable with that observed in cultured HeLa cells (supplemental Fig. S1C, available at www.jneurosci.org as supplemental material) and to that previously reported in COS cells transfected with GFP-tagged TIA-1 (Kedersha et al., 2000).

The ability of the N-terminal portion of Pum2 to induce SG formation and to recruit TIAR after overexpression prompted us to further investigate whether a specific domain was responsible for the observed effect. The Pum2 protein contains a poly Q (glutamine)-rich region (QRR) located upstream of the PHD (supplemental Fig. S2A, available at www.jneurosci.org as supplemental material). Because this domain was present in the Pum_1–651 construct, we tested whether a smaller part of the N-terminal half containing the QRR domain was still sufficient for the induction of SGs. Two different constructs of the N terminus were therefore created, the first containing amino acids 1–226 (Pum2_1–226) in which no relevant domains or amino acid sequences have been identified, and the second (Pum2_225–651) containing the QRR region. The mere overexpression of full-length Pum2 leads to SG formation in HeLa cells, whereas Pum2_1–226 displayed a homogenous cellular distribution (Fig. 9Ai). This region was not recruited into SGs even during arsenite-induced metabolic stress (data not shown). Pum2_1–651 induces the formation of two distinct populations of aggregates: large cytoplasmic granules that recruited TIAR and small foci that were visible within the cytoplasm and the nucleus that did not recruit TIAR (Fig. 9Aii and inset). In contrast, expression of Pum2_225–651 only induced small foci (Fig. 9Aiii). Because these small aggregates were reminiscent of P-bodies because of their size and shape, we immunostained transfected HeLa cells for both TIAR and Lsm1 (Fig. 9B,C). As shown in neurons, the overexpression of Pum2_FL induced the formation of TIAR-positive SGs. Although Pum2 and TIAR colocalized, Lsm1 was consistently excluded. Interestingly, the overexpression of Pum2_225–651 induced the formation of small aggregates that caused an accumulation of TIAR in the absence of metabolic stress. However, the Pum2_225–651 aggregates did not overlap with the TIAR granules but were adjacent to them. They also remained excluded from P-bodies (Fig. 9C).

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Figure 9.

A glutamine-rich region in the N terminus is responsible for the formation of SGs after overexpression. N-terminal deletion mutants (see scheme) of Pum2–EYFP were transiently transfected into HeLa cells. A, The N terminus lacking the QRR in the N terminus (1–226; Ai) does not form SGs after overexpression or in the presence of arsenite (data not shown). The portion of the N terminus containing the QRR forms small puncta and causes the formation of TIAR-positive SGs (225–651; Aiii). These small puncta colocalize with the TIAR granules (enlargements marked with asterisks). B, P-bodies detected with an anti-Lsm1 antibody (red) are adjacent to but not overlapping with either overexpressed Pum2_FL (green) or TIAR (blue). C, The small granules induced by overexpression of Pum2_225–651 (green) do not colocalize with either Lsm1 (red) or TIAR (blue). Arrowheads indicate small Pum2 granules. Arrows indicate Lsm1-positive P-bodies.Scale bar, 10 μm.

We then determined whether the overexpression of Pum2_FL and Pum2_225–651 led to the recruitment of other components of SGs by staining HeLa with eIF4E, PABP, and TIA-1 (Fig. 10). As expected, Pum2_FL overexpression formed aggregates containing all SG markers. Similarly, the overexpression of Pum2_225–651 caused a rearrangement of TIA-1, eIF4E, and PABP in a similar manner to that of TIAR. PABP, another known component of SGs (Kedersha et al., 2005), was also recruited to these structures in neurons treated with arsenite (supplemental Fig. S3B, available at www.jneurosci.org as supplemental material).

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Figure 10.

Overexpression of Pum2 recruits additional components of SGs. Overexpression of both Pum2_FL and the N terminus containing the QRR (Pum2_225–651) in HeLa cells causes the aggregation of TIA1, eIF4E, and PABP in a pattern similar to TIAR. The * and ** symbols represent enlargements in color. Scale bar, 10 mm.

Pum2 containing SGs are in dynamic equilibrium with polysomes

During stress, polysomes and SGs are in a state of dynamic equilibrium (Kedersha et al., 2005). Cycloheximide interferes with translation by blocking peptide elongation and halting ribosomes on translating mRNA (Kedersha et al., 2000). This drug has been used to demonstrate the dynamic nature of SGs in cell lines. After addition of cycloheximide, SGs are no longer visible when cells are stained with antibodies used for SG detection. With polysomes no longer available for recruitment into SGs, any existing granules will dissolve and escape visualization. We demonstrate a similar phenomenon in neuronal SGs. Cycloheximide led to the loss of Pum2-positive SGs in both the soma and dendrites. This was also observed with SGs induced by overexpression of Pum2_FL. Conversely, puromycin, by releasing polysomes from translating mRNA, leads to the formation of SGs in the absence of stress (Kedersha et al., 2000).

Arsenite is known to increase the number of P-bodies along with SGs (Kedersha et al., 2005). P-bodies are sites of mRNA decay (Sheth and Parker, 2003), and we have shown that during stress, the number of P-bodies increases in dendrites. This indicates that in the presence of stress, the potential exists for dendritically localized mRNA to be targeted for decay. Although it has been proposed that SGs and P-bodies communicate via molecules such as CPEB (Wilczynska et al., 2005), it remains to be demonstrated that mRNA targeted for decay passes through SGs before delivery to P-bodies. We observed no colocalization of SG and P-body markers under any of our experimental conditions.

A glutamine-rich region in the N terminus is capable of inducing SG assembly

Overexpression of the Pum2 QRR induced the formation of small aggregates that recruited known components of SGs such as TIAR, TIA-1, PABP, and eIF4E. Although visibly similar to the smaller P-bodies, Pum2 QRR aggregates were not associated with these structures. Rather, these small aggregates clustered around the periphery of SGs. Similar effects have been reported with the overexpression of a glutamine-rich region of TIA-1. The TIA-1 glutamine-rich domain, similar to that of Pum2, is structurally related to prion protein (Gilks et al., 2004). It has therefore been proposed that TIA-1 prion-like activity is responsible for the initial protein aggregation during stress. This leads to the formation of a scaffold onto which stalled initiation complexes and other proteins associated with SGs can bind (Gilks et al., 2004). Unlike TIA-1 and TIAR, which are primarily nuclear proteins, Pum2 is found in distal dendrites. Prion-like activity of Pum2 could therefore mediate the aggregation of nontranslating RNPs into dendritic SGs. What remains to be resolved is the catalytic event that would induce Pum2 prion-like activity. TIA-1 prion-like behavior is regulated by levels of heat shock protein 70 (HSP70) protein in a complex relationship. High levels of HSP70 reduce or even block aggregation of TIA-1 (Gilks et al., 2004). However, our preliminary data show that overexpression of Pum2 does not induce upregulation or downregulation of HSP70 translation (J. P. Vessey and P. Macchi, unpublished observations).