The Phosphorylation of CCR6 on Distinct Ser/Thr Residues in the Carboxyl Terminus Differentially Regulates Biological Function.

CCR6 is a G protein-coupled receptor (GPCR) that recognizes a single chemokine ligand, CCL20 and is primarily expressed by leukocytes. Upon ligand binding, CCR6 activates Gαi heterotrimeric G proteins to induce various potential cellular outcomes through context-specific cell signaling. It is well known that differential phosphorylation of Ser and Thr residues in the C-terminal domains or intracellular loops of GPCRs can generate barcodes that regulate GPCR function by regulating the recruitment of ß-arrestins. In this study, we demonstrate that ligand binding to CCR6 induces receptor phosphorylation at Ser/Thr residues in the C-terminal tail, rather than intracellular loops. Using mutagenesis experiments, we determined that distinct clusters of Ser/Thr residues in the C-terminal domain differentially regulate CCL20-induced signaling and cellular response. Substituting the Thr360/Ser361/Thr363 cluster or the Ser370/Ser371 cluster with Ala residues modulated cellular response upon CCL20 stimulation. Notably, receptor internalization, chemotaxis, F-actin distribution, transient ERK1/2 activation, and ß-arrestin 2 recruitment were oppositely affected by mutating the two clusters, suggesting that phosphorylation of CCR6 C-terminal Ser/Thr residues directs the cell signaling response upon receptor activation. Moreover, activated CCR6 weakly recruited ß-arrestin 1 in comparison with ß-arrestin 2, and the two arrestin proteins seemed to play overlapping but distinct roles in mediating CCL20/CCR6-induced cellular responses. Taken together, the effects of site-specific Ser/Thr phosphorylation on CCR6 demonstrate the existence of barcodes on the protein that dictate the activation of different cell signaling profiles and lead to distinct biological outcomes.

The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.

The signaling adaptor MAVS forms prion-like aggregates to activate an innate antiviral immune response after viral infection. However, the molecular mechanisms that regulate MAVS aggregation are poorly understood. Here we identified TRIM31, an E3 ubiquitin ligase of the TRIM family of proteins, as a regulator of MAVS aggregation. TRIM31 was recruited to mitochondria after viral infection and specifically regulated antiviral signaling mediated by RLR pattern-recognition receptors. TRIM31-deficient mice were more susceptible to infection with RNA virus than were wild-type mice. TRIM31 interacted with MAVS and catalyzed the Lys63 (K63)-linked polyubiquitination of Lys10, Lys311 and Lys461 on MAVS. This modification promoted the formation of prion-like aggregates of MAVS after viral infection. Our findings reveal new insights in the molecular regulation of MAVS aggregation and the cellular antiviral response through TRIM31-mediated K63-linked polyubiquitination of MAVS.

Enhanced diacylglycerol production by phospholipase D activation is responsible for abnormal increase in concanavalin A cap formation in polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mice.

We previously reported that enhanced ceramide production induces calpain-mediated proteolysis of protein kinase C (PKC) in leukocytes from Chediak-Higashi syndrome (CHS). In the present study, we demonstrated that phospholipase D (PLD) inhibitors ameliorated abnormal increases in concanavalin A (Con A) cap formation in polymorphonuclear leukocytes (PMNs) from beige mouse, an animal model of CHS. PLD activity in PMNs from beige mice enhanced at 30 to 60s after Con A stimulation. In Con A-stimulated beige PMNs, both neutral sphingomyelinase (N-SMase) and acidic sphingomyelinase (A-SMase) activities enhanced, and ceramide levels are also increased. We found that ceramide levels were reversed by the treatment of beige PMNs with propranolol which inhibits phosphatidic acid phosphohydrolase. In addition, we showed that diacylgycerol (DAG) analogs enhance both N-SMase and A-SMase activities in PMNs from normal mice. We subsequently examined the association of CHS1 with PLD, and showed that expression of a truncated mutant of CHS1 in 293T cells induced abnormally rapid activation of PLD after phorbol ester stimulation. Moreover, we showed that specific inhibitors of 14-3-3 proteins, which interact with CHS1/LYST and bind PKC, did not affect abnormal increases in Con A cap formation in beige PMNs. These results suggest that the enhanced DAG production via the PLD pathway is associated with abnormal increases in Con A cap formation in beige PMNs, and that CHS1 may be involved in the regulation of PLD activity.

Tight coupling between nucleus and cell migration through the perinuclear actin cap.

Although eukaryotic cells are known to alternate between 'advancing' episodes of fast and persistent movement and 'hesitation' episodes of low speed and low persistence, the molecular mechanism that controls the dynamic changes in morphology, speed and persistence of eukaryotic migratory cells remains unclear. Here, we show that the movement of the interphase nucleus during random cell migration switches intermittently between two distinct modes - rotation and translocation - that follow with high fidelity the sequential rounded and elongated morphologies of the nucleus and cell body, respectively. Nuclear rotation and translocation mediate the stop-and-go motion of the cell through the dynamic formation and dissolution, respectively, of the contractile perinuclear actin cap, which is dynamically coupled to the nuclear lamina and the nuclear envelope through LINC complexes. A persistent cell movement and nuclear translocation driven by the actin cap are halted following the disruption of the actin cap, which in turn allows the cell to repolarize for its next persistent move owing to nuclear rotation mediated by cytoplasmic dynein light intermediate chain 2.

Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance.

Neutrophil infiltration during inflammation is regulated by PILRα via modulation of integrin activation.

Acute inflammatory responses are important in host defense, whereas dysregulated inflammation results in life-threatening complications. Here we found that paired immunoglobulin-like type 2 receptor alpha (PILRα), an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), negatively regulated neutrophil infiltration during inflammation. Pilra(-/-) mice had increased neutrophil recruitment to inflammatory sites and were highly susceptible to endotoxin shock. Pilra(-/-) neutrophils showed enhanced transmigration ability and increased adhesion to the ß(2) integrin ligand ICAM-1. PILRα expressed on neutrophils constitutively associated in cis with its ligands, resulting in clustering of PILRα during stimulation with a chemoattractant. Clustering of PILRα enhanced ITIM-mediated signaling, thus modulating ß(2) integrin inside-out activation. These data demonstrate that neutrophil recruitment in inflammatory responses is regulated by PILRα via modulation of integrin activation.

Nestin is not essential for development of the CNS but required for dispersion of acetylcholine receptor clusters at the area of neuromuscular junctions.

Nestin is expressed in many different progenitors during development including those of the CNS, heart, skeletal muscle, and kidney. The adult expression is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction, and renal podocytes. In addition, this intermediate filament protein has served as a marker of neural stem/progenitor cells for close to 20 years. Therefore it is surprising that its function in development and adult physiology is still poorly understood. Here we report that nestin deficiency is compatible with normal development of the CNS. The mutant mice, however, show impaired motor coordination. Furthermore, we found that the number of acetylcholine receptor clusters, the nerve length, and the endplate bandwidth are significantly increased in neuromuscular junction area of nestin-deficient mice. This is similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5), a candidate downstream affecter of nestin. Moreover, we demonstrate that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knock-outs, suggesting that the observed nestin deficiency phenotype is the consequence of aberrant Cdk5 activity.

Reduced postsynaptic GABAA receptor number and enhanced gaboxadol induced change in holding currents in Purkinje cells of the dystrophin-deficient mdx mouse.

UNLABELLED: Duchenne muscular dystrophy (DMD) is caused by the absence of a functional transcript of the protein dystrophin. DMD is associated with a range of cognitive deficits that are thought to result from a lack of the protein dystrophin in brain structures involved in cognitive functions. The CNS involvement extends to an impairment of cognitive abilities, with many DMD boys having significant reduction in IQ. In the cerebellum, dystrophin is normally localized at the postsynaptic membrane of GABAergic synapses on Purkinje cells. Here, we investigate the effect of an absence of dystrophin on the number of GABA(A) channels located at the synapse in cerebellar Purkinje cells of the dystrophin-deficient mdx mouse. Whole-cell patch-clamp recordings of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were performed in cerebellar slices from mdx and littermate control mice. Our results showed that the number of receptors at GABAergic synapses in the cerebellar Purkinje cell was significantly reduced in mdx mice (38.38 ± 2.95) compared to littermate controls (53.03 ± 4.11). Furthermore, when gaboxadol was added to the bath, the change in holding current in mdx mice was significantly enhanced (65.01 ± 5.89pA) compared to littermate controls (37.36 ± 3.82pA). The single channel unitary conductance and the rise and decay time of mIPSCs were not significantly different in these two groups of mice, indicating that those GABA(A) channels located at the postsynaptic sites in the mdx mice function normally. CONCLUSION: There is a reduction in the number of functional receptors localized at GABAergic synapses in the cerebellar Purkinje cells of dystrophin-deficient mdx mice and an increase in a gaboxadol induced holding current, which is evidence for an increase in extrasynaptic GABA(A) receptors in mdx mice. We hypothesize that the absence of dystrophin, from mdx Purkinje cells, reduces the number of post-synaptic GABA(A) receptors and as a result there is an increase in extrasynaptic receptors. If similar changes occur in the CNS in boys with DMD, it will impact on the function of neural networks and may contribute to some of the motor, behavioral and cognitive impairment apparent in many boys with DMD.

Complex phosphorylation dynamics control the composition of the Syk interactome in B cells.

Spleen tyrosine kinase Syk provides critical transducer functions for a number of immune cell receptors and has been implicated in the generation of several forms of leukemias. Catalytic activity and the ability of Syk to interact with other signaling elements depend on the phosphorylation status of Syk. We have now identified and quantified the full spectrum of phosphoacceptor sites in human Syk as well as the interactome of Syk in resting and activated B cells by high-resolution mass spectrometry. While the majority of inducible phosphorylations occurred on tyrosine residues, one of the most frequently detected phosphosites encompassed serine 297 located within the linker insert distinguishing the long and short isoforms of Syk. Full-length Syk can associate with more than 25 distinct ligands including the 14-3-3γ adaptor protein, which binds directly to phosphoserine 297. The latter complex attenuates inducible plasma membrane recruitment of Syk, thereby limiting antigen receptor-proximal signaling pathways. Collectively, the established ligand library provides a basis to understand the complexity of the Syk signaling network.

TLR-mediated signaling pathways circumvent the requirement for DAP12 in mast cells for the induction of inflammatory mediator release.

TLR, expressed on the surface of mast cells, respond to a variety of bacterial and viral components to induce and enhance high-affinity IgE receptor-mediated cytokine production. Recent reports have indicated that specific TLR-dependent responses in macrophages and dendritic cells are regulated by the ITAM-containing molecule, DAP12. When phosphorylated, DAP12 recruits Syk, which is a critical molecule for mast cell activation. We therefore examined whether DAP12 similarly regulates TLR-mediated responses in mast cells. DAP12 was confirmed to be expressed in both human and mouse mast cells and, upon phosphorylation, to recruit Syk. However, although TLR agonists induced cytokine production, and synergistically enhanced high-affinity IgE receptor-mediated cytokine production, surprisingly, they failed to increase DAP12 phosphorylation in mouse bone marrow-derived mast cells (BMMC). Furthermore, normal TLR-mediated responses were observed in DAP12(-/-) BMMC. However, DAP12 phosphorylation and subsequent Syk recruitment were observed in BMMC following Con A-induced aggregation of mannose-glycosylated receptors, and these responses, together with Con A-induced degranulation, were substantially reduced in the DAP12(-/-) BMMC. These data demonstrate that TLR have differential requirements for DAP12 for their function in different cell types and that the inability of TLR to influence mast cell degranulation may be linked to their inability to utilize DAP12 to recruit Syk.

MHC class I and TCR avidity control the CD8 T cell response to IL-15/IL-15Rα complex.

IL-15 operates via a unique mechanism termed transpresentation. In this system, IL-15 produced by one cell type is bound to IL-15Rα expressed by the same cell and is presented to apposing cells expressing the IL-15Rß/γC complex. We have shown that administering soluble IL-15Rα complexed with IL-15 can greatly enhance IL-15 activity. We now show that the naive CD8 T cell response to exogenous IL-15/IL-15Rα complex is MHC class I dependent. In the absence of ß2 microglobulin, naive CD8 T cells scarcely proliferated in response to IL-15/IL-15Rα complex, whereas memory cells proliferated, although to a lesser extent, compared with levels in control mice. The loss of ß2m or FcRn slightly reduced the extended half-life of IL-15/IL-15Rα complex, whereas FcRn deficiency only partially reduced the naive CD8 T cell proliferative response to IL-15/IL-15Rα complex. In addition, we demonstrated a link between TCR avidity and the ability of a T cell to respond to IL-15/IL-15Rα complex. Thus, T cells expressing low-avidity TCR responded poorly to IL-15/IL-15Rα complex, which correlated with a poor homeostatic proliferative response to lymphopenia. The inclusion of cognate peptide along with complex resulted in enhanced proliferation, even when TCR avidity was low. IL-15/IL-15Rα complex treatment, along with peptide immunization, also enhanced activation and the migratory ability of responding T cells. These data suggest that IL-15/IL-15Rα complex has selective effects on Ag-activated CD8 T cells. Our findings have important implications for directing IL-15/IL-15Rα complex-based therapy to specific Ag targets and illustrate the possible adjuvant uses of IL-15/IL-15Rα complex.

Syndecan-1 couples the insulin-like growth factor-1 receptor to inside-out integrin activation.

Syndecan-1 (Sdc1) engages and activates the αvß3 (and/or αvß5) integrin when clustered in human carcinoma and endothelial cells. Although the engagement is extracellular, the activation mechanism is cytoplasmic. This talin-dependent, inside-out signaling pathway is activated downstream of the insulin-like growth factor-1 receptor (IGF1R), whose kinase activity is triggered by Sdc1 clustering. In vitro binding assays using purified receptors suggest that association of the Sdc1 ectodomain with the integrin provides a 'docking face' for IGF1R. IGF1R docking and activation of the associated integrin is blocked by synstatin (SSTN(92-119)), a peptide derived from the integrin engagement site in Sdc1. IGF1R colocalizes with αvß3 integrin and Sdc1 in focal contacts, but fails to associate with or activate the integrin in cells either lacking Sdc1 or expressing Sdc1(Δ67-121), a mutant that is unable to form the Sdc1-integrin-IGF1R ternary complex. Integrin activation is also blocked by IGF1R inhibitors or by silencing IGF1R or talin expression with small-interfering RNAs (siRNAs). In both cases, expression of the constitutively active talin F23 head domain rescues integrin activation. We recently reported that SSTN(92-119) blocks angiogenesis and impairs tumor growth in mice, therefore this Sdc1-mediated integrin regulatory mechanism might be a crucial regulator of disease processes known to rely on these integrins, including tumor cell metastasis and tumor-induced angiogenesis.

Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 exvivo expansion.

Peripheral blood natural killer (NK) cell therapy for acute myeloid leukemia has shown promise in clinical trials after allogeneic stem cell transplantation. Cord blood (CB) is another potentially rich source of NK cells for adoptive immune therapy after stem cell transplantation. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. However, despite expressing normal surface activating and inhibitory NK receptors, CB-derived NK cells have poor cytolytic activity. In this study, we investigate the cellular mechanism and demonstrate that unmanipulated CB-NK cells exhibit an impaired ability to form F-actin immunologic synapses with target leukemia cells compared with peripheral blood-derived NK cells. In addition, there was reduced recruitment of the activating receptor CD2, integrin leukocyte function-associated antigen-1, and the cytolytic molecule perforin to the CB-NK synapse site. Exvivo interleukin (IL)-2 expansion of CB-NK cells enhanced lytic synapse formation including CD2 and leukocyte function-associated antigen-1 polarization and activity. Furthermore, the acquired antileukemic function of IL-2-expanded CB-NK cells was validated using a nonobese diabetic severe combined immunodeficient IL-2 receptor common γ-chain null mouse model. We believe our results provide important mechanistic insights for the potential use of IL-2-expanded CB-derived NK cells for adoptive immune therapy in leukemia.

Postsynaptic development of the neuromuscular junction in mice lacking the gamma-subunit of muscle nicotinic acetylcholine receptor.

The mammalian muscle nicotinic acetylcholine receptor (AChR) is composed of five membrane-spanning subunits and its composition differs between embryonic and adult muscles. In embryonic muscles, it is composed of two alpha-, one beta-, one delta-, and one gamma-subunit; the gamma-subunit is later replaced by the epsilon-subunit during postnatal development. This unique temporal expression pattern of the gamma-subunit suggests it may play specific roles in embryonic muscles. To address this issue, we examined the formation and function of the neuromuscular junction in mouse embryos deficient in the gamma-subunit. At embryonic day 15.5, AChR clusters were absent and the spontaneous miniature endplate potentials were undetectable in the mutant muscles. However, electrical stimulation of the nerves triggered muscle contraction and elicited postsynaptic endplate potential (EPP) in the mutant muscles, although the magnitude of the muscle contraction and the amplitudes of the EPPs were smaller in the mutant compared to the wild-type muscles. Reintroducing a wild-type gamma-subunit into the mutant myotubes restored the formation of AChR clusters in vitro. Together, these results have demonstrated that functional AChRs were present in the mutant muscle membrane, but at reduced levels. Thus, in the absence of the gamma-subunit, a combination of alpha, beta, and delta subunits may assemble into functional receptors in vivo. These results also suggest that the gamma-subunit maybe involved in interacting with rapsyn, a cytoplasmic protein required for AChR clustering.

HSP90 beta regulates rapsyn turnover and subsequent AChR cluster formation and maintenance.

Rapsyn, an acetylcholine receptor (AChR)-interacting protein, is essential for synapse formation at the neuromuscular junction (NMJ). Like many synaptic proteins, rapsyn turns over rapidly at synapses. However, little is known about molecular mechanisms that govern rapsyn stability. Using a differential mass-spectrometry approach, we identified heat-shock protein 90beta (HSP90beta) as a component in surface AChR clusters. The HSP90beta-AChR interaction required rapsyn and was stimulated by agrin. Inhibition of HSP90beta activity or expression, or disruption of its interaction with rapsyn attenuated agrin-induced formation of AChR clusters in vitro and impaired the development and maintenance of the NMJ in vivo. Finally, we showed that HSP90beta was necessary for rapsyn stabilization and regulated its proteasome-dependent degradation. Together, these results indicate a role of HSP90beta in NMJ development by regulating rapsyn turnover and subsequent AChR cluster formation and maintenance.

LRP4 serves as a coreceptor of agrin.

Neuromuscular junction (NMJ) formation requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. We report that LRP4, a low-density lipoprotein receptor (LDLR)-related protein, is expressed specifically in myotubes and binds to neuronal agrin. Its expression enables agrin binding and MuSK signaling in cells that otherwise do not respond to agrin. Suppression of LRP4 expression in muscle cells attenuates agrin binding, agrin-induced MuSK tyrosine phosphorylation, and AChR clustering. LRP4 also forms a complex with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations indicate that LRP4 is a coreceptor of agrin that is necessary for MuSK signaling and AChR clustering and identify a potential target protein whose mutation and/or autoimmunization may cause muscular dystrophies.

Impaired TCR signaling through dysfunction of lipid rafts in sphingomyelin synthase 1 (SMS1)-knockdown T cells.

During T cell activation, TCRs cluster at the center of the T cell-antigen-presenting cell interface forming the central supramolecular activation cluster. Although it has been suggested that sphingolipid- and cholesterol-rich microdomains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals, an actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has not been reported. After cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1, we established a SM-knockdown cell line (Jurkat-SMS1/kd) by transfection of SMS1-short-interfering RNA into Jurkat T cells, which is deficient in membrane expression of SM. Upon CD3 stimulation, expression of CD69 (the earliest leukocyte activation antigen), activation-induced cell adhesion and proliferation as well as TCR clustering was severely impaired in Jurkat-SMS1/kd cells. CD3-induced tyrosine phosphorylation and association of linker for activation of T cell with ZAP-70 and Grb2 and phosphorylation of protein kinase C (PKC) were also severely impaired in Jurkat-SMS1/kd cells. Finally, translocation of TCR, ZAP-70 and PKC into lipid rafts was markedly decreased in Jurkat-SMS1/kd cells. These findings indicate that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.

Despite GABAergic neurotransmission, GABAergic innervation does not compensate for the defect in glycine receptor postsynaptic aggregation in spastic mice.

In the hypoglossal nucleus of wild-type mice, early mixed glycinergic-GABAergic inhibitory transmission becomes mainly glycinergic during postnatal maturation. In spastic mice (SPA), a model of human hyperekplexic syndrome, an insertion into the gene of the glycine receptor (GlyR) beta subunit results in a decreased accumulation of GlyRs at postsynaptic sites and an impaired glycinergic neurotransmission. In SPA mice displaying a mild phenotype (B6C3Fe strain), a compensatory process involving an increased aggregation of GABA(A) receptors (GABA(A)Rs) at postsynaptic sites was proposed to explain survival of mutant animals until adulthood. However, C57BL/6J strain SPA mice which express a lower amount of GlyR beta subunit die 2-3 weeks after birth, suggesting that GABAergic compensation does not necessarily take place. We performed a morphofunctional study of inhibitory synapses in the developing hypoglossal nucleus of C57BL/6J SPA mice. In this mutant, the inhibitory synaptic activity was mainly GABAergic. Accordingly, we observed a developmental loss of glycinergic presynaptic terminals and an increase in the density of GABAergic presynaptic terminals during the first two postnatal weeks. In addition, while C57BL/6J SPA mice displayed a strong impairment in GlyR aggregation at postsynaptic loci, the proportion of inhibitory presynaptic terminals facing diffuse GABA(A)Rs significantly increased during development. Our results suggest crosstalk between postsynaptic and presynaptic elements, leading to the developmental regulation of the presynaptic terminal neurotransmitter content according to the level of postsynaptic GlyR aggregation. They also indicate that GABAergic neurotransmission does not compensate for defects in GlyR postsynaptic aggregation leading to spastic syndrome in C57BL/6J SPA mice.

Retrograde regulation of motoneuron differentiation by muscle beta-catenin.

Synapse formation requires proper interaction between pre- and postsynaptic cells. In anterograde signaling, neurons release factors to guide postsynaptic differentiation. However, less is known about how postsynaptic targets retrogradely regulate presynaptic differentiation or function. We found that muscle-specific conditional knockout of beta-catenin (Ctnnb1, also known as beta-cat) in mice caused both morphologic and functional defects in motoneuron terminals of neuromuscular junctions (NMJs). In the absence of muscle beta-catenin, acetylcholine receptor clusters were increased in size and distributed throughout a wider region. Primary nerve branches were mislocated, whereas secondary or intramuscular nerve branches were elongated and reduced in number. Both spontaneous and evoked neurotransmitter release was reduced at the mutant NMJs. Furthermore, short-term plasticity and calcium sensitivity of neurotransmitter release were compromised in beta-catenin-deficient muscle. In contrast, the NMJ was normal in morphology and function in motoneuron-specific beta-catenin-deficient mice. Taken together, these observations indicate a role for muscle beta-catenin in presynaptic differentiation and function, identifying a previously unknown retrograde signaling in the synapse formation and synaptic plasticity.

Gephyrin clustering is required for the stability of GABAergic synapses.

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transsynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.