Previous studies have implicated the actin-binding proteins spectrin and Coracle in NMJ development and in the selective clustering of GluR subunits most likely through the regulation of pre- and postsynaptic F-actin (Chen et al., 2005
; Pielage et al., 2006
). Here we identify an additional novel mechanism by which postsynaptic F-actin/spectrin is regulated during NMJ expansion. Previously, we reported that the aPKC-Baz-Par-6 complex is present at the NMJ and that aPKC was required for normal NMJ expansion and presynaptic microtubule stability (Ruiz-Canada et al., 2004
). Here, we show that aPKC and Baz in conjunction with the lipid and protein phosphatase PTEN play an additional role in regulating postsynaptic F-actin/spectrin through a novel mechanism. We propose that Baz is required to stabilize the postsynaptic F-actin/spectrin meshwork, and this process involves changes in its phosphorylation state (). Our evidence suggests that proper targeting of Baz to the postsynaptic region requires its phosphorylation by aPKC, but its retention at this region may requires its dephosphorylation by PTEN ().
Proposed model for the regulation of the synaptic cytoskeleton by the aPKC-Baz-Par-6 complex at the larval NMJ
Several lines of evidence support this model. First, reduction of aPKC, Baz and PTEN levels in the muscles result in a similarly dramatic reduction in postsynaptic F-actin. Second, Baz localization at the postsynaptic F-actin-rich region depends on its phosphorylation state. While phosphorylated Baz is excluded, dephosphorylated Baz is concentrated at this site. Third, aPKC is also excluded from the F-actin-rich region and a decrease in Baz phosphorylation by aPKC results in a decrease in Baz at the F-actin rich area. This suggests that aPKC-dependent phosphorylation of Baz occurs outside the F-actin-rich region and that this phosphorylation may be required for proper targeting but not for retention of Baz at the actin-rich postsynaptic region. Fourth, PTEN colocalizes with dephospho-Baz at the actin-rich postsynaptic region and reducing PTEN levels results in a reduction in Baz at this site, and in an increase in phospho-Baz levels in the surrounding area. Thus, the retention of Baz at the F-actin-rich area requires its dephosphorylation, which is likely mediated through PTEN. Fifth, there is a significant overlap in NMJ phenotypes upon downregulating Baz, aPKC and PTEN, and Baz and PTEN interact genetically suggesting that they might function in the same pathway during NMJ expansion.
Studies in mammalian cells have implicated Par-3 in the maintenance of actin-based junctions, such as the tight junction (Chen and Macara, 2005
; Nishimura et al., 2005
). Baz is an aPKC substrate, and our studies at the NMJ show that it is in exact colocalization with F-actin. Collectively, this indicates that the effects of aPKC on F-actin/spectrin are mediated most likely through Baz, and that Baz is required for the normal organization of the postsynaptic F-actin/spectrin meshwork. Any reduction in postsynaptic Baz levels as seen by decreasing aPKC, Baz, or PTEN resulted in a decrease in the density and thickness of the postsynaptic F-actin cytoskeleton.
Several potential mechanisms by which Par-3/Baz might regulate F-actin have been suggested, including dephosphorylation of Cofilin through the inhibition of LIM kinase (Chen and Macara, 2006
), and regulation of the Rac Guanine nucleotide exchange factor, Tiam1 (Chen and Macara, 2005
; Nishimura et al., 2005
). Although, Drosophila
LIMK and Tiam1 are absent from the postsynaptic region (Sone et al., 1997
; Eaton and Davis, 2005
), Baz might regulate dPIX, another Rho-GEF that is enriched postsynaptically (Parnas et al., 2001
) and its downstream effector dPAK (Conder et al., 2007
). Another potential mechanism could involve an interaction between the PDZ2 domain of Baz and membrane phospholipids (Wu et al., 2007
). By binding to phospholipids, Baz might bring together a number of actin regulators to the membrane to remodel postsynaptic F-actin.
Cell culture studies demonstrate that Par-3 is phosphorylated by aPKC, which results in the dissociation of the two proteins and targeting and stabilization of phospho-Par-3 at sites of tight junction formation (Hirose et al., 2002
; Nagai-Tamai et al., 2002
). We hypothesize that phosphorylation of Baz at S980 is similarly required to target Baz to the postsynaptic region. We determined that phospho-S980-Baz was distributed in puncta at the muscle cortex, but excluded from the postsynaptic F-actin-rich area. Consistent with previous studies (Nagai-Tamai et al., 2002
), altering muscle aPKC levels resulted in the respective increase or decrease in phospho-Baz levels in the muscle. Thus, our immunocytochemical results, supported by our biochemical assays, strongly validate the idea that Baz is phosphorylated by aPKC in the muscle. To our knowledge, this is the first study to demonstrate the phosphorylation of Baz by aPKC in the context of an intact organism, and during NMJ development. We suggest that, as in mammals, phosphorylation of Baz might disrupt the binding between Baz and aPKC allowing phospho-Baz to be mobilized to the postsynaptic region. However, unlike previous studies which hypothesized that phospho-Par-3 was stabilized at the apical region (Nagai-Tamai et al., 2002
), our studies showed that phospho-Baz was absent from the postsynaptic region. Instead, we found that Baz at this region was present in a dephosphorylated state. Thus we postulate that while targeting Baz to the postsynaptic region requires aPKC phosphorylation, its actual retention at this site necessitates its dephosphorylation.
Studies in epithelial cells show that Baz interacts with the lipid and protein phosphatase PTEN through binding between the PDZ2-3 domain of Baz and the PDZ binding motif in PTEN2 (von Stein et al., 2005
; Pinal et al., 2006
). Further, these studies demonstrated that Baz was required for the recruitment of PTEN to regions of actin remodeling (Pinal et al., 2006
). These observations made PTEN a prime candidate for Baz dephosphorylation. Confirming this model, we found that PTEN was colocalized with dephospho-Baz at the postsynaptic region. Moreover, reducing PTEN activity resulted in a decrease in dephospho-Baz at the postsynaptic region and an increase in phospho-Baz in muscle suggesting that PTEN-dependent dephosphorylation of Baz is necessary to retain Baz at the postsynaptic region. Additionally, our biochemical assays indicate that this dephosphorylation requires the PDZ interaction between PTEN and Baz and is mediated by the protein phosphatase activity of PTEN. PTEN has been shown to directly dephosphorylate many different proteins such as FAK kinase (Tamura et al., 1998
), but might also function indirectly through the activation of other protein phosphatases (Traweger et al., 2008
). Notably, we found that the localization of Baz and PTEN was interdependent for their mutual localization at the postsynaptic region. Consistent with results of previous studies (Pinal et al., 2006
), Baz downregulation also decreased postsynaptic PTEN supporting the idea that Baz is required for PTEN localization at postsynaptic sites.
Several studies have implicated PTEN in regulating synaptic structure and function (Fraser et al., 2008
), neurotransmitter receptors (Ning et al., 2004
; Ji et al., 2006
), hippocampal LTD (Wang et al., 2006
), neuronal arborization, and social interactions in mice (Kwon et al., 2006
). Our studies demonstrate for the first time that PTEN is involved in inducing the dephosphorylation of Baz and the regulation of the postsynaptic F-actin cytoskeleton. Thus, these studies reveal a potential molecular mechanism for PTEN function in the nervous system.
A previous study in embryonic NMJs demonstrated a role for postsynaptic F-actin in the proper clustering of GluRIIA-, but not of GluRIIB-receptors through the fly Band 4.1 homolog, Coracle (Cora), which appears to interact directly with the C-terminal tail of GluRIIA (Chen et al., 2005
). In cora
mutants, GluRIIA-, but not GluRIIB-cluster size and function was reduced (Chen et al., 2005
). Pharmacological disruption of F-actin using Lantrunculin mimicked this mutant phenotype, leading the authors to suggest that Cora might function to directly anchor GluRIIA subunits to F-actin (Chen et al., 2005
). In our studies and Ruiz-Canada et al (2004)
we found that GluRIIA-, GluRIIB-, and GluRIII-cluster volume and intensity was significantly increased upon downregulating either aPKC, Baz, or PTEN, and that this phenotype was accompanied by an increase in the amplitude of mEJPs. However, in our studies the postsynaptic F-actin region was not completely disrupted, but significantly reduced in size. This raises the interesting possibility that beyond anchoring receptors on the postsynaptic membrane, the F-actin and spectrin domain might act as a barrier to the diffusion and clustering of receptors at the postsynaptic area or may affect the recycling of receptors. In this regard, it is important to note that an increase in GluRIIA, GluRIIB, and GluRIII size was also observed in another study where spectrin was dowregulated exclusively in the larval muscles using RNAi (Pielage et al., 2006
). Furthermore, this study also found an increase in the mEJP amplitude upon downregulation of spectrin in the muscle. These studies implicated spectrin in regulating active zone size and spacing, as well as synaptic efficacy. While some of the phenotypes reported upon spectrin elimination in the muscle, were similar to those examined here upon downregulating aPKC, Baz, and PTEN, others were quite different. For example, spectrin elimination resulted in disrupted SSR and abnormal localization of DLG. The loss of SSR, in addition to being a result of disorganized Dlg, as suggested by the study (Pielage et al., 2006
), could also be due to a change in postsynaptic F-actin configuration from meshwork to wisps seen in spec
mutants and ϐ-Spec-RNAi-post. In contrast, we found that the SSR was intact upon expressing aPKC RNAi in muscles, and SSR markers such as DLG and Scrib were not affected upon downregulating aPKC, Baz, and PTEN. It was suggested that either the F-actin/spectrin network might be responsible for the stabilization of GluRs, the spectrin-actin hexagonal lattice might serve as a framework that constrains the size of active zones and postsynaptic receptors, or that changes in GluR size and spacing could be a secondary consequence of the disruption of the SSR (Pielage et al., 2006
). Our findings that the SSR in aPKC-RNAi-post and DLG distribution upon downregulating aPKC, Baz, and PTEN is not affected, but that GluR clusters are increased in size suggest that the size of GluR cluster is not necessarily dependent on the SSR or DLG. Together with the studies of Chen et al. (2006)
demonstrating that disruption of F-actin does not alter the formation of GluRIIB-clusters, the results of Pielage et al (2006)
and ours are consistent with the idea that the F-actin/spectrin network might restrict GluR- cluster size.
Downregulating aPKC, Baz, or PTEN levels in the muscle, besides decreasing postsynaptic F-actin localization, also result in a substantial increase in synaptic bouton size and a decrease in NMJ expansion. As the larval NMJ arbors are completely surrounded by the muscle membrane, NMJ expansion and bouton size are likely regulated by forces involving a balance between cytoskeletal extension at the presynaptic arbors and cytoskeletal retraction at the postsynaptic sites. Not surprisingly, interfering with this balance leads to misregulation of NMJ extension and bouton size. Interestingly, our previous studies suggest that aPKC might play different roles in the regulation of the presynaptic versus postsynaptic cytoskeletons. At the presynapse, aPKC is associated with microtubules and regulates microtubule stability through interactions with the presynaptic MAPB-related protein Futsch (Ruiz-Canada et al., 2004
). In muscles, aPKC is also associated with microtubules, but regulates both postsynaptic F-actin/spectrin through Baz (this report) and postsynaptic microtubules (Ruiz-Canada et al., 2004
). The exact mechanisms for the regulation of postsynaptic microtubules by aPKC are not known. This regulation might involve the modulation of an as yet unknown muscle microtubule-associated protein, or might result from indirect regulation by the stabilization of F-actin, which might exclude microtubules. In summary, our study reveals a novel mechanism by which the postsynaptic F-actin cytoskeleton is regulated during NMJ growth. In this mechanism F-actin organization at the postsynaptic region depends at least in part on Baz function. In turn, proper localization of Baz at this F-actin region depends on the opposing actions of aPKC and PTEN. The conservation of the aPKC-Baz-PTEN interaction across different cell types argues in favor of common mechanisms for cytoskeletal regulation by this complex in many tissues. Further, our studies establish a mechanism by which the dynamics of the postsynaptic actin cytoskeleton might be regulated during plasticity in the brain.