Scribble Interacts with β-Catenin and Localizes at Synapses
scribble has been implicated in the development of synapse structure and function (Roche et al., 2002
). The mammalian homolog of scribble has a primary structure similar to that of its fly homolog (Santoni et al., 2002
), and localizes to distinct regions in the brain, including the hippocampus (http://www.brain-map.org/
). However the function of scribble in neurons remains unknown. To determine the distribution of scribble in hippocampal neurons, 10 DIV primary hippocampal cultured neurons were immunostained for scribble. Scribble immunoreactivity was observed throughout the cell, being localized to both dendrites and axons (Supplementary Figure S1). Previous work has demonstrated that scribble is localized to synapses in cultured cerebellar neurons (Audebert et al., 2004
; Takizawa et al., 2006
). Analysis of hippocampal cultures demonstrates a similar synaptic distribution for scribble (, A–D), whereby 84.8% of excitatory synapses, identified by colocalization of the presynaptic vesicular glutamate transporter, VGLUT-1, and the excitatory postsynaptic marker, PSD-95, were associated with scribble immunoreactive puncta (). Scribble puncta that are not associated with VGLUT-1 and PSD-95 may represent populations at inhibitory synapses or at nonsynaptic sites (, B and D, arrows). Moreover, the majority of synaptophysin, an integral synaptic vesicle (SV) marker, colocalizes with scribble (, E–G, ). Scribble-positive sites that are not associated with synaptophysin may represent mobile scribble clusters or scribble clusters that are at nascent synapses before the recruitment of synaptic vesicles.
Figure 1. Scribble localizes to synapses. (A–M) Confocal images of 12 DIV hippocampal cultures. (H–M) To determine the subcellular localization of fluorescently tagged proteins in individual neurons, cells were transfected at <1% efficiency (more ...)
Analysis of scribble localization at synapses
GFP-tagged synaptic vesicle marker proteins have been widely used to mark presynaptic sites in vertebrates and do not compromise the secretory physiology of the synapse (Sankaranarayanan and Ryan, 2000
). Moreover, our work has previously demonstrated that the pattern of synaptophysin-GFP (Syn-GFP) expression is comparable to that of endogenous SV proteins (Bamji et al., 2003
). Endogenous scribble puncta were found to colocalize with Syn-GFP to approximately the same degree, further demonstrating the faithful use of this tagged marker (, H–J, ). To determine whether RFP-conjugated human scribble (RFP-hScrib) is appropriately expressed, neurons were cotransfected with RFP-hScrib and Syn-GFP. The percent colocalization of RFP-hScrib and Syn-GFP was highly similar to that observed with endogenous scribble and synaptophysin (, K–M, ). Finally, the density of RFP-hScrib clusters was similar to that of endogenous scribble (, p > 0.05), and RFP-hScrib was found associated with endogenous synaptophysin clusters (data not shown). Thus, our data indicate that Syn-GFP and RFP-hScrib are faithful markers for assaying appropriate localization patterns of the corresponding endogenous proteins.
We next examined the association of endogenous scribble and β-catenin in neurons. Immunohistochemical analysis demonstrated that a large proportion of β-catenin puncta colocalize with scribble puncta. These colocalized clusters were highly enriched at bassoon immunoreactive sites, suggesting that scribble colocalizes with β-catenin at synapses (, A–E, ). To further demonstrate that scribble associates with β-catenin at synapses, crude synaptosomal fractions from E18 brains were prepared and immunoprecipitated with antibodies against scribble and β-catenin. Coimmunoprecipitation assays demonstrate that scribble associates with β-catenin and cadherin at synapses (F). The interaction between β-catenin and scribble was further confirmed using GST pulldown assays (G).
Figure 2. Scribble associates with β-catenin at synapses. (A–E) Confocal images of 12 DIV hippocampal cultures demonstrating colocalization of scribble, β-catenin, and the presynaptic marker, bassoon. Higher magnifications of the inset from (more ...)
β-Catenin consists of three domains: an N-terminal domain that interacts with α-catenin, a central domain of 12 armadillo repeats that binds to cadherin and LEF/TCF transcription factors (Daniels et al., 2001
; Ivanov et al., 2001
), and a C-terminal domain that interacts with transcriptional regulators and contains a PDZ-binding motif (Perego et al., 2000
). Our previous work demonstrated that the PDZ-binding motif is essential for the clustering of SVs at synaptic junctions (Bamji et al., 2003
). To investigate whether this domain is important for the interaction between β-catenin and scribble, β-catenin deletion mutant lacking the PDZ-binding motif (GST-β-catΔPDZb) was generated (G). Full-length β-catenin (GST-β-cat FL) was able to pulldown scribble and cadherin from synaptosomal fractions. In contrast, deletion of the C-terminal 10 amino acids containing the PDZ-binding motif abolished the association between β-catenin and scribble. As expected, GST-β-catΔPDZb was able to pulldown cadherin from synaptosomal fractions (G). Together, this demonstrates that scribble associates with cadherin and β-catenin at synapses via the PDZ-binding motif of β-catenin.
SVs Are Mislocalized in Hippocampal Neurons Lacking Scribble
To study the role of scribble at synapses, scribble protein levels were attenuated in hippocampal neurons using RNAi. To minimize possible “off-target” effects, two shRNAs (RNAi-1 and -2) and one cocktail of three siRNA oligonucleotides (RNAi-3) were used. The efficacy of the RNAis was examined using Western blot analysis. Although the efficiency of transfection was on average only 35.9 ± 2%, a significant decrease in scribble protein levels was observed in cultures transiently transfected with RNAi-1, -2, or -3, compared with cultures transfected with control RNAi (RNAi-C; , A and B). In contrast, β-catenin levels remained similar in scribble RNAi-expressing cells (A). (It is important to note that B represents raw data and has not been normalized for transfection efficiency.) Similarly, quantitative RT-PCR revealed significant decreases in scribble transcripts in cultures transfected with RNAi (Supplementary Figure S2). The efficiency of scribble depletion was similar among all three RNAi treatments and was used interchangeably in subsequent experiments. To further confirm the knockdown of scribble protein in neurons expressing RNAi, cultured hippocampal neurons were transfected with either RNAi-C or each one of the three RNAis and immunolabeled for scribble. Cells were cotransfected with Syn-GFP to mark RNAi-transfected neurons. At low magnifications, expression levels of scribble in cell bodies of control cells were abundant (C, D asterisks), whereas in cells expressing RNAi-1, somatic scribble levels were dramatically decreased (, E and F, asterisks). This was consistently observed in neurons expressing each of the three RNAis, indicating an effective knockdown of scribble in neuronal cultures.
Figure 3. RNAi-mediated knockdown of scribble protein levels in primary neurons. (A and B) Neurons were transfected with control RNAi (RNAi-C), or three distinct scribble RNAis (RNAi-1-3) with ~35% transfection efficiency using the Amaxa nucleofector system. (more ...)
Interestingly, the pattern of Syn-GFP expression in RNAi-expressing neurons was altered compared with control neurons. In wild-type cells, discrete Syn-GFP puncta were observed along the axon (D′ and , A and B). In contrast, in scribble RNAi-expressing cells, discrete Syn-GFP puncta were not observed. (F′ and , F and G). To confirm the distribution of SVs in RNAi-transfected cells, cultures were immunostained with synaptotagmin, another integral SV marker. Synaptotagmin expression appeared similar to that of Syn-GFP, with fewer large immunopositive clusters along RNAi-expressing axons (, J–L).
Figure 4. SVs are more diffusely distributed along the axon in neurons expressing scribble RNAi constructs. (A–J) Confocal images of 10 DIV hippocampal neurons cotransfected with Syn-GFP plus scribble RNAis using Lipofectamine 2000 (<1% transfection (more ...)
To further investigate the pattern of SV localization in neurons, and specifically its localization at synaptic sites, cells were transfected with Syn-GFP and RNAi-C or each of the three RNAis and immunostained with bassoon and PSD-95 to label pre- and postsynaptic sites, respectively. In control neurons, the fluorescence intensity of the Syn-GFP–positive puncta was high, as observed in the pseudocolored, low-magnification image (A) and in the intensity distribution histogram (K). Discrete fluorescence intensity peaks representing individual Syn-GFP puncta were observed, and the level of fluorescence intensity between peaks (interpunctal intensity) was relatively low (, A and K). These Syn-GFP–positive puncta were localized to synapses as observed by its colocalization with PSD-95 and bassoon (, B–E). In contrast, in RNAi-expressing cells, the fluorescence intensity distribution of Syn-GFP was relatively uniform along the axon, with an overall increase in basal intensity compared with the interpunctal intensity of wild-type cells (, F and L). Although Syn-GFP fluorescence was more diffusely distributed along the axon, PSD-95 and bassoon colocalization (synapses) was still evident in these cells (, G–J).
We next measured synaptophysin levels to ensure that changes in the pattern of Syn-GFP distribution in scribble knockdown cells were not due to overall changes in synaptophysin expression. Both synaptophysin and β-catenin levels remained similar in scribble RNAi-expressing cells compared with control (M), suggesting that attenuation of scribble perturbs the distribution of SV clusters, while leaving the overall expression of SV proteins undisturbed.
Variability in the “punctate-ness” of Syn-GFP was observed in RNAi-expressing cells. To determine the distribution of vesicles along the axon, the Feret's diameter (defined as the greatest distance possible between any two points along the boundary of a region of interest, hereafter called the “length”) of the Syn-GFP fluorescence signal was determined as described previously (Bamji et al., 2003
). To avoid bias, all transfected neurons on each coverslip were imaged and quantified. A 28.6–34.4% increase was observed in the average length of Syn-GFP fluorescence signal in knockdown cells compared with wild-type. Although the increase was significant, the difference between wild-type and knockdown cells was minimized due to an increased density of small Syn-GFP puncta in knockdown cells which we believe arise from small clusters of SVs that are not retained at synapses. To address this, the sum of the length of Syn-GFP fluorescence signal per 10 μm axon length (hereafter referred to as the “coverage”) was used to represent the distribution of SVs along the axon (N). No significant difference was seen in Syn-GFP coverage between neurons expressing Syn-GFP alone and Syn-GFP plus RNAi-C. In contrast, the coverage of Syn-GFP along the axon was over twofold greater in RNAi-expressing neurons compared with neurons expressing RNAi-C. The magnitude of the phenotype was similar for all three RNAis. Importantly, coexpression of RFP-hScrib that is insensitive to our RNAis rescued this phenotype, indicating that the observed effects were primarily due to specific interference with scribble function (N).
To further assess the distribution of SVs at synapses, the intensity of Syn-GFP fluorescence at synapses (defined here as points of colocalization between PSD-95 and bassoon) was quantified. A significant decrease in Syn-GFP fluorescence intensity at synapses was observed in RNAi-transfected cells compared with control cells (O), suggesting that SV number is specifically diminished at synaptic junctions upon scribble knockdown. Synapse number was also quantified by counting the density of bassoon-positive puncta that colocalized with PSD-95 in control and RNAi-transfected cells, and no significant difference was observed (P).
To determine the role of scribble on presynaptic development beyond its role in SV localization, the expression pattern of the presynaptic cytoskeletal matrix protein, bassoon, was examined. Bassoon is recruited to synapses in large dense-core vesicles along with other components of the active zone, including piccolo, N-cadherin, syntaxin, SNAP-25, and chromogranin B, independently of the vesicles that transport SV proteins to synapses (Zhai et al., 2001
). The density, size, and intensity of endogenous bassoon was similar between control and scribble RNAi-expressing neurons (Supplementary Figure S3). These data suggest that scribble is involved in some, but not all steps, of synapse assembly.
Taken together, our results demonstrate an important role for scribble in localizing SVs to synapses. In cells expressing scribble RNAi, discrete SV clusters are rarely observed, and the intensity of Syn-GFP fluorescence at synapses significantly decreases. Interestingly, scribble knockdown does not appear to affect the localization of other presynaptic proteins such as bassoon or the density of synapses along the axon. This phenotype is very similar to the observation in β-catenin knockout neurons (Bamji et al., 2003
β-Catenin Localizes Scribble to Synapses
Our previous work has demonstrated a role for β-catenin in the localization of SVs (Bamji et al., 2003
). In light of our above results showing that scribble is also required for normal localization of SVs and that scribble and β-catenin exist in complex with each other, we next determined whether scribble acts in concert with β-catenin to localize SVs. First, the localization of β-catenin in scribble knockdown neurons was examined. In wild-type cells, endogenous β-catenin displayed a punctate expression pattern and colocalized with Syn-GFP (, A–C). In scribble knockdown cells, there was a diffuse pattern of Syn-GFP expression; however the β-catenin remained punctate (, D–F). Moreover, the density of β-catenin along the axon, as well as the area of β-catenin, remained similar in control and scribble RNAi-transfected cells (, G and H). This suggested that the localization of β-catenin at synapses is not dependent on scribble.
Figure 6. β-Catenin localization is not affected in scribble RNAi-expressing cells. (A–F) Confocal images of 10 DIV hippocampal neurons transfected with Syn-GFP and RNAi-C (A–C) or RNAi-3 (D–F) using Lipofectamine 2000 (<1% (more ...)
We next tested the role of β-catenin in the recruitment and synaptic localization of scribble. Hippocampal neurons prepared from B6.129-Ctnnb1tm2Kem
/KnwJ mice (homozygous β-catenin
flox mice) were transfected with a vector expressing the Cre recombinase to ablate β-catenin
. This methodology has previously been shown to efficiently ablate β-catenin
in vitro (Bamji et al., 2003
). In control cells, RFP-hScrib was expressed in a punctate pattern and colocalized with Syn-GFP and PSD-95 at synapses (, A–D). In contrast, expression of the Cre recombinase in β-catenin flox
neurons resulted in the expected diffuse pattern of Syn-GFP expression, as well as a diffuse pattern of RFP-hScrib expression (, E–H). Indeed, the average length of RFP-hScrib fluorescence along the axon was significantly greater in cells lacking β-catenin (O). Interestingly, PSD-95 puncta at postsynaptic sites apposed to transfected axons remained punctate (G).
Figure 7. Scribble is diffusely distributed long the axon in cells lacking β-catenin. (A–H) Hippocampal neurons cultured from 10 DIV B6.129-Ctnnb1tm2Kem/KnwJ (homozygous β-catenin flox) mice were cotransfected using Lipofectamine 2000 (<1% (more ...)
We have demonstrated that the PDZ-binding motif of β-catenin is important for the interaction between β-catenin and scribble (G). To test whether the PDZ-binding motif is important for the synaptic localization of scribble, neurons were transfected with either β-cat FL or β-catΔPDZb, plus Syn-GFP and RFP-hScrib. In cells expressing β-cat FL, Syn-GFP and RFP-hScrib had a punctate distribution and were largely colocalized, similar to that observed with endogenous synaptophysin and scribble proteins (, I–K, and , E–G). As previously demonstrated, cells expressing β-catΔPDZb exhibited a diffuse pattern of Syn-GFP expression (L; Bamji et al., 2003
). Interestingly, in these cells, scribble was also diffusely distributed along the axon (, M and P). These data suggest that β-catenin plays an important role in localizing scribble to synaptic sites and that scribble acts downstream of β-catenin to localize SVs.