Fig 5 - uploaded by Joav Prives
Content may be subject to copyright.
Dominant interfering Rho inhibits the ability of laminin to cluster AChR. (A) In order to assess the contribution of Rho activation to laminin-induced AChR clustering, differentiated muscle cells were microinjected with RhoN19 or vector. After microinjection, the cells were treated with laminin (10 nM) overnight. One day after injection, the effects of AChR surface distribution in RhoN19-expressing (c,d) versus vector-expressing cells (a,b) treated with laminin were examined by confocal microscopy of cultures surface-labeled with TMR-Bgt. Those myotubes injected with RhoN19 were identified with co-injected FITC-goat anti-mouse antibody (a,c). Myotubes expressing RhoN19 did not display full-sized AChR clusters after treatment with laminin (d), compared with those myotubes injected with vector only (b). Scale bar, 10 m. (B) Quantitative comparison of the number of AChR clusters on the surface of myotubes expressing the Rho mutant versus control myotubes clearly documents the inhibiting effect of the dominant interfering RhoN19 on AChR cluster (black bars) and microcluster (grey bars) formation in response to laminin (10 nM) treatment (error bars represent ±s.e.m.; n=40 cells from five or more separate platings).
Source publication
During neuromuscular junction formation, extracellular matrix-mediated signals cause muscle surface acetylcholine receptors (AChRs) to aggregate at synaptic sites. Two extracellular matrix proteins, agrin and laminin, have each been shown to initiate signaling pathways that culminate in AChR clustering in cultured muscle cells. Here we present evid...
Similar publications
The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requiremen...
We previously reported that the activation of prostaglandin E receptor EP3 subtype caused neurite retraction via small GTPase
Rho in the EP3B receptor-expressing PC12 cells (Katoh, H., Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem.271, 29780–29784). However, a potential downstream effector of Rho that induces neurite retraction was not identif...
Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin-2 receptor activates small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), and dominant-negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and...
Rho-GTPase has been implicated in axon outgrowth. However, not all of the critical steps controlled by Rho have been well characterized. Using cultured cerebellar granule neurons, we show here that stromal cell-derived factor (SDF)-1alpha, a neural chemokine, is a physiological ligand that can turn on two distinct Rho-dependent pathways with opposi...
Tumor recurrence after liver transplantation still remains a significant problem in patients with hepatocellular carcinoma. The small GTPase Rho/Rho-associated kinase (ROCK) pathway is involved in the motility and invasiveness of cancer cells. We investigated whether tacrolimus activated the Rho/ROCK signal pathway to promote the invasiveness of ra...
Citations
... It is known, for instance, that laminin binding causes DG and syntrophin to be tyrosinephosphorylated. The latter, notably, has the effect of increasing syntrophin's binding to Grb2, which, through forming a complex with Sos1, activates Rac1 and PAK1-JNK, ultimately resulting in the remodeling of the actin cytoskeleton, activation of the PI3K/AKT pathway, and the aggregation of AChRs (Langenbach and Rando 2002;Marangi et al. 2002;Sadasivam et al. 2005;Sotgia et al. 2003;Spence et al. 2004;Weston et al. 2007;Zhou et al. 2007;Zhou et al. 2006). ...
In astrocytes, the water-permeable channel aquaporin-4 (AQP4) is concentrated at the endfeet that abut the blood vessels of the brain. The asymmetric distribution of this channel is dependent on the function of dystroglycan (DG), a co-expressed laminin receptor, and its associated protein complex. We have demonstrated that the addition of laminin to astrocytes in culture causes the clustering of AQP4, DG, and lipid rafts. The last, in particular, have been associated with the initiation of cell signaling. As laminin binding to DG in muscle cells can induce the tyrosine phosphorylation of syntrophin and laminin requires tyrosine kinases for acetylcholine receptor clustering in myotubes, we asked if signal transduction might also be involved in AQP4 clustering in astrocytes. We analyzed the timecourse of AQP4, DG, and monosialotetrahexosylganglioside (GM1) clustering in primary cultures of rat astrocytes following the addition of laminin, and determined that the clustering of DG precedes that of AQP4 and GM1. We also showed that laminin induces the formation of phosphotyrosine-rich clusters and that the tyrosine kinase inhibitor, genistein, disrupts the laminin-induced clustering of both β-DG and AQP4. Using the Kinexus antibody microarray chip, we then identified protein-serine kinase C delta (PKCδ) as one of the main proteins exhibiting high levels of tyrosine phosphorylation upon laminin treatment. Selective inhibitors of PKC and siRNA against PKCδ disrupted β-DG and AQP4 clustering, and also caused water transport to increase in astrocytes treated with laminin. Our results demonstrate that the effects of laminin on AQP4 localization and function are relayed, at least in part, through PKC signaling.
... It is possible that when drebrin association with actin is changed, it impacts the interactions of other actin-binding proteins. It was shown that drebrin competes for binding to F-actin with cofilin and αactinin, and inhibits the actin filament severing and cross-linking, respectively [92][93][94]. Drebrin downregulation might result in increased actin severing by cofilin and release actin-bound proteins, such as beta-catenin, enabling its translocation to the nucleus. Moreover, changes in the conformation of actin filaments caused by drebrin modify the preferential binding of other actin-organizing proteins to different actin cytoskeletal structures [95,96]. ...
Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.
... More recent findings show that agrin-dependent RhoA activation requires a progressive inactivation of Rac1 (Bai et al., 2018) that, in turn, is necessary for the formation of full-size nAChR clusters (Weston et al., 2003;Weston et al., 2000). In addition, in vitro studies have shown that RhoA activity is required for the laminin-dependent aggregation of nAChRs on myotubes (Weston et al., 2007). ...
The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor axon and a skeletal muscle fibre that allows muscle contraction and the coordinated movement in many species. A main hallmark of the mature NMJ is the assembly of nicotinic acetylcholine receptor (nAChR) aggregates in the muscle postsynaptic domain, that distributes in perfect apposition to presynaptic motor terminals. To assemble its unique functional architecture, initial embryonic NMJs undergo an early postnatal maturation process characterised by the transformation of homogenous nAChR-containing plaques to elaborate and branched pretzel-like structures. In spite of a detailed morphological characterisation, the molecular mechanisms controlling the intracellular scaffolding that organises a postsynaptic domain at the mature NMJ have not been fully elucidated. In this review, we integrate evidence of key processes and molecules that have shed light on our current understanding of the NMJ maturation process. On the one hand, we consider in vitro studies revealing the potential role of podosome-like structures to define discrete low nAChR-containing regions to consolidate a plaque-to-pretzel transition at the NMJ. On the other hand, we focus on in vitro and in vivo evidence demonstrating that members of the Ras homologous (Rho) protein family of small GTPases (small Rho GTPases) play indispensable roles on NMJ maturation by regulating the stability of nAChR aggregates. We combine this evidence to propose that small Rho GTPases are key players in the assembly of podosome-like structures that drive the postsynaptic maturation of vertebrate NMJs.
... MACF1 plays an important role in maintaining synaptic differentiation and efficient synaptic transmission in mice (Oury et al., 2019). Actin and actin-associated proteins involved in F-actin polymerization, such as cortactin and an Arp2/3 complex, are enriched at nAChR clusters in cultured muscle cells (Weston et al., 2007) and are activated by MuSK. The precise mechanisms involved remain poorly understood. ...
Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles. The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction, the nicotinic acetylcholine receptor. Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse. This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis. The review also covers the application of a powerful biophysical technique, superresolution optical microscopy, to image the nicotinic receptor in live cells and follow its motional dynamics.
The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor, as observed in myasthenia gravis.
... Transmission of impulses from motoneurons to myofibers takes place in highly specialized chemical synapses called NMJs. Knowing that laminins are crucial for NMJ formation and clustering of postsynaptic acetylcholine receptor (AChR) [60][61][62] and that Pax7 influence laminin synthesis, we analyzed the expression of genes coding laminins involved in the formation of NMJs and innervation of myofibers. To test whether impaired formation of NMJs in Pax7−/− teratomas is associated with specific laminins, we estimated the expression of mRNAs encoding laminin alpha 4 (Lama4), laminin alpha 5 (Lama5), and laminin beta 2 (Lamb2) subunits. ...
... The laminins are heterodimers of alpha, beta, and gamma subunit. Interestingly, within the NMJs, the postsynaptic membrane is rich in laminin chains alpha 2, alpha 4, alpha 5, beta 2, and gamma 1 [60][61][62][63]. Interactions between the abovementioned laminins are necessary for proper AChR anchorage and NMJ maturation during embryonic development (summarized in [71]). ...
Background:
Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population.
Methods:
In vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers.
Results:
We determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7-/- teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7-/- teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7-/- ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7-/- teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7-/- teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7-/- teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7-/- teratomas was impaired.
Conclusions:
Pax7-/- ESCs differentiate in vivo to embryonic myoblasts more readily than Pax7+/+ cells. In the absence of functional Pax7, initiation of myogenic differentiation is facilitated, and as a result, the expression of mesoderm embryonic myoblast markers is upregulated. However, in the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7-/- ESCs during advanced stages of myogenesis.
... It functions in cell attachment and mobility. It is also known to function in a complex that activates Rho GTPases [37]. ...
We present a novel method for the identification of sets of mutually exclusive gene alterations in a given set of genomic profiles. We scan the groups of genes with a common downstream effect on the signaling network, using a mutual exclusivity criterion that ensures that each gene in the group significantly contributes to the mutual exclusivity pattern. We test the method on all available TCGA cancer genomics datasets, and detect multiple previously unreported alterations that show significant mutual exclusivity and are likely to be driver events.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0612-6) contains supplementary material, which is available to authorized users.
... LAMA2, the alpha subunit of laminin, functions in cell attachment and mobility. It is also known to function in a complex that activates Rho GTPases [31]. SPTB is a member of the spectrin family, which are membrane cytoskeletal proteins that functions in cell membrane organization and stability. ...
Recent cancer genome studies have identified numerous genomic alterations in cancer genomes. It is hypothesized that only a fraction of these genomic alterations drive the progression of cancer -- often called driver mutations. Current sample sizes for cancer studies, often in the hundreds, are sufficient to detect pivotal drivers solely based on their high frequency of alterations. In cases where the alterations for a single function are distributed among multiple genes of a common pathway, however, single gene alteration frequencies might not be statistically significant. In such cases, we expect to observe that most samples are altered in only one of those alternative genes because additional alterations would not convey an additional selective advantage to the tumor. This leads to a mutual exclusion pattern of alterations, that can be exploited to identify these groups. We developed a novel method for the identification of sets of mutually exclusive gene alterations in a signaling network. We scan the groups of genes with a common downstream effect, using a mutual exclusivity criterion that makes sure that each gene in the group significantly contributes to the mutual exclusivity pattern. We have tested the method on all available TCGA cancer genomics datasets, and detected multiple previously unreported alterations that show significant mutual exclusivity and are likely to be driver events.
... Therefore, Wnts may act via similar pathways that are activated by Agrin. For example, Wnt3 regulation of Agrininduced clustering does not require ROCK, but Rac1 (Henriquez et al., 2008), a Rho GTPase that is necessary for Agrin-induced clustering (Weston et al., 2000;Weston et al., 2003;Weston et al., 2007). ...
Neuromuscular junction (NMJ) is a cholinergic synapse where motor neurons elicit muscle contraction. Agrin and its coreceptors LRP4 and MuSK are critical for vertebrate NMJ formation. This paper reviews recent evidence for Wnts and Wnt signaling molecules in NMJ formation including a possible retrograde mechanism by muscle β-catenin. We also present data that Wnt3a, 7a, 8a and 10b could inhibit agrin-mediated AChR clustering. Together with the stimulating effect of Wnt9a, 9b, 10b, 11 and 16 on AChR clustering in the absence of agrin, these results suggest diverse roles for Wnt ligands in NMJ development.
... 64,65 ECM molecules, such as fibronectin, laminin, vitronectin and collagen, have all been implicated in JNK activation. [66][67][68][69] Recently, a new concept of integrin cycling was described in endothelial cells, where the dynamic and constant formation of new FAs required newly engaged integrins in order to fully activate the JNK pathway. 70 In addition, it has been observed that JNK signaling could be activated by mechanical strain 71,72 and fluid shear stress through phosphorylation of FAK at Tyr-397. ...
c-Jun N-terminal Kinase (JNK) is a family of protein kinases, which are activated by stress stimuli such as inflammation, heat stress and osmotic stress, and regulate diverse cellular processes including proliferation, survival and apoptosis. In this review, we focus on a recently discovered function of JNK as a regulator of intercellular adhesion. We summarize the existing knowledge regarding the role of JNK during the formation of cell-cell junctions. The potential mechanisms and implications for processes requiring dynamic formation and dissolution of cell-cell junctions including wound healing, migration, cancer metastasis and stem cell differentiation are also discussed.
... Indeed, a study on cultured myotubes from an SMA patient revealed a decrease in AChR clustering in response to soluble agrin administration (Arnold et al., 2004). Key effectors downstream from MuSK activation are Rho GTPases, which reorganise the actin cytoskeleton to initiate redistribution of AChRs (Weston et al., 2007). Given the previous data reporting disrupted actin dynamics in response to reduced Smn levels, this pathway may be of particular interest. ...
Spinal muscular atrophy (SMA) is a devastating childhood motor neuron disease caused by mutations and deletions within the survival motor neuron 1 (SMN1) gene. Although other tissues may be involved, motor neurons remain primary pathological targets, with loss of neuromuscular junctions (NMJs) representing an early and significant event in pathogenesis. Although defects in axonal outgrowth and pathfinding have been observed in cell culture and in lower organisms upon Smn depletion, developmental defects in mouse models have been less obvious. Here, we have employed the Smn(2B/-) mouse model to investigate NMJ remodelling during SMA pathology, induced reinnervation, and paralysis. We show that whilst NMJs are capable of remodelling during pathogenesis, there is a marked reduction in paralysis-induced remodelling and in the nerve-directed re-organisation of acetylcholine receptors. This reduction in remodelling potential could not be attributed to a decreased rate of axonal growth. Finally, we have identified a loss of terminal Schwann cells which could contribute to the defects in remodelling/maintenance observed. Our work demonstrates that there are specific defects in NMJ remodelling in an intermediate SMA mouse model, which could contribute to or underlie pathogenesis in SMA. The development of strategies that can promote the remodelling potential of NMJs may therefore be of significant benefit to SMA patients.
