RESUMEN
The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.
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Proteómica , Sinapsis , Adolescente , Animales , Niño , Preescolar , Humanos , Lactante , Recién Nacido , Ratones , Adulto Joven , Cognición/fisiología , Espinas Dendríticas , Edad Gestacional , Macaca , Neuronas/metabolismo , Densidad Postsináptica/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Especificidad de la Especie , Sinapsis/metabolismo , Sinapsis/fisiologíaRESUMEN
Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.
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Adhesiones Focales , Cinesinas , Microtúbulos , Factores de Intercambio de Guanina Nucleótido Rho , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Cinesinas/metabolismo , Cinesinas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Miosina Tipo II/metabolismo , Talina/metabolismo , Talina/genética , AnimalesRESUMEN
Enzymes or enzyme complexes can be concentrated in different cellular loci to modulate distinct functional processes in response to specific signals. How cells condense and compartmentalize enzyme complexes for spatiotemporally distinct cellular events is not well understood. Here we discover that specific and tight association of GIT1 and ß-Pix, a pair of GTPase regulatory enzymes, leads to phase separation of the complex without additional scaffolding molecules. GIT1/ß-Pix condensates are modular in nature and can be positioned at distinct cellular compartments, such as neuronal synapses, focal adhesions, and cell-cell junctions, by upstream adaptors. Guided by the structure of the GIT/PIX complex, we specifically probed the role of phase separation of the enzyme complex in cell migration and synapse formation. Our study suggests that formation of modular enzyme complex condensates via phase separation can dynamically concentrate limited quantities of enzymes to distinct cellular compartments for specific and optimal signaling.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Animales , Proteínas de Ciclo Celular/química , Proteínas Activadoras de GTPasa/química , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Moleculares , Paxillin/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.
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Proteínas Portadoras , Proteínas de la Membrana , Terminales Presinápticos , Receptores de GABA-A , Sinapsis , Ácido gamma-Aminobutírico , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ácido gamma-Aminobutírico/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genéticaRESUMEN
Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I-dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-ß expression in macrophages. Generation of Arhgef2(-/-) mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.
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Inmunidad Innata/inmunología , Microtúbulos/inmunología , ARN Viral/inmunología , Factores de Intercambio de Guanina Nucleótido Rho/inmunología , Transducción de Señal/inmunología , Animales , Células COS , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Expresión Génica/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Inmunidad Innata/genética , Immunoblotting , Virus de la Influenza A/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1 , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microtúbulos/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal/genéticaRESUMEN
The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.
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Proteínas de Unión al GTP rho , Proteína de Unión al GTP rhoB , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Aparato de Golgi/metabolismo , Endosomas/metabolismoRESUMEN
Fibronectin (FN)-binding integrins control a variety of cellular responses through Rho GTPases. The FN-binding integrins, αvß3 and α5ß1, are known to induce different effects on cell morphology and motility. Here, we report that FN-bound αvß3 integrin, but not FN-bound α5ß1 integrin, triggers the dissociation of the RhoA GEF Lfc (also known as GEF-H1 and ARHGEF2 in humans) from microtubules (MTs), leading to the activation of RhoA, formation of stress fibres and maturation of focal adhesions (FAs). Conversely, loss of Lfc expression decreases RhoA activity, stress fibre formation and FA size, suggesting that Lfc is the major GEF downstream of FN-bound αvß3 that controls RhoA activity. Mechanistically, FN-engaged αvß3 integrin activates a kinase cascade involving MARK2 and MARK3, which in turn leads to phosphorylation of several phospho-sites on Lfc. In particular, S151 was identified as the main site involved in the regulation of Lfc localization and activity. Our findings indicate that activation of Lfc and RhoA is orchestrated in FN-adherent cells in an integrin-specific manner.
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Integrina alfa5beta1 , Integrina alfaV , Humanos , Integrina alfaV/metabolismo , Integrina alfa5beta1/metabolismo , Integrinas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosforilación , Proteína de Unión al GTP rhoA/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismoRESUMEN
Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-ß pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.
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Transdiferenciación Celular , Dexametasona , Glaucoma , Miofibroblastos , Factores de Intercambio de Guanina Nucleótido Rho , Malla Trabecular , Dexametasona/farmacología , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismo , Malla Trabecular/citología , Transdiferenciación Celular/efectos de los fármacos , Animales , Humanos , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/citología , Ratones , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Glaucoma/patología , Glaucoma/metabolismo , Células Cultivadas , Glucocorticoides/farmacología , Ratones Endogámicos C57BL , MasculinoRESUMEN
A cell's size affects the likelihood that it will die. But how is cell size controlled in this context and how does cell size impact commitment to the cell death fate? We present evidence that the caspase CED-3 interacts with the RhoGEF ECT-2 in Caenorhabditis elegans neuroblasts that generate "unwanted" cells. We propose that this interaction promotes polar actomyosin contractility, which leads to unequal neuroblast division and the generation of a daughter cell that is below the critical "lethal" size threshold. Furthermore, we find that hyperactivation of ECT-2 RhoGEF reduces the sizes of unwanted cells. Importantly, this suppresses the "cell death abnormal" phenotype caused by the partial loss of ced-3 caspase and therefore increases the likelihood that unwanted cells die. A putative null mutation of ced-3 caspase, however, is not suppressed, which indicates that cell size affects CED-3 caspase activation and/or activity. Therefore, we have uncovered novel sequential and reciprocal interactions between the apoptosis pathway and cell size that impact a cell's commitment to the cell death fate.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Actomiosina/metabolismo , Animales , Apoptosis/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caspasas/genética , Caspasas/metabolismo , Tamaño de la Célula , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismoRESUMEN
Neurons are vulnerable to physical insults, which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used the PVD neurons in C. elegans with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites, and fusion among the higher-order branches as reported before. We quantified the regeneration pattern into three aspects-territory length, number of branches, and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signalling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidylserine (PS) exposure/PS in dendrite regeneration. We found that neither dendrite regrowth nor fusion was affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase, CED-10 and its upstream GEF, TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, the function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteínas de Unión al GTP rac , Animales , Axones/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Regeneración Nerviosa/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.
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Proteínas del Tejido Nervioso , Displasia Retiniana , Factores de Intercambio de Guanina Nucleótido Rho , Animales , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Displasia Retiniana/genética , Displasia Retiniana/metabolismo , Displasia Retiniana/patología , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismoRESUMEN
Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes (NLGN1-3, 4X, and 4Y). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules.SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission.
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Quinasa 5 Dependiente de la Ciclina , Sinapsis , Animales , Ratones , Ratas , Moléculas de Adhesión Celular/metabolismo , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Fosforilación/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Serina/metabolismo , Sinapsis/metabolismo , Transmisión SinápticaRESUMEN
RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-ß1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.
Asunto(s)
Fibrosis , Factores de Intercambio de Guanina Nucleótido Rho , Factor de Transcripción Sp1 , Transactivadores , Proteína de Unión al GTP rhoA , Animales , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transactivadores/metabolismo , Transactivadores/genética , Ratones , Ratas , Retroalimentación Fisiológica , Masculino , Ratones Endogámicos C57BL , Humanos , Transducción de Señal , Porcinos , Fosforilación , Modelos Animales de Enfermedad , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Obstrucción Ureteral/genética , Ratas Sprague-Dawley , Línea Celular , Factores de TranscripciónRESUMEN
The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gßγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C ß3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.
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Actinas , Ácidos Araquidónicos , Estructuras de la Membrana Celular , Neoplasias , Receptores Eicosanoides , Humanos , Actinas/metabolismo , Neoplasias/metabolismo , Toxina del Pertussis/farmacología , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Estructuras de la Membrana Celular/metabolismo , Nanotubos , Receptores Eicosanoides/antagonistas & inhibidores , Receptores Eicosanoides/metabolismo , Línea Celular Tumoral , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Transducción de SeñalRESUMEN
AIMS: Tau is a key player in Alzheimer's disease (AD) and other Tauopathies. Tau pathology in the brain directly correlates with neurodegeneration in AD. The recent identification of a rapid variant of AD demands an urgent need to uncover underlying mechanisms leading to differential progression in AD. Accordingly, we aimed to dissect the underlying differential mechanisms of toxicity associated with the Tau protein in AD subtypes and to find out subtype-dependent biomarkers and therapeutic targets. METHODS: To identify and characterise subtype-specific Tau-associated mechanisms of pathology, we performed comparative interactome mapping of Tau protein in classical AD (cAD) and rapidly progressive AD (rpAD) cases using co-immunoprecipitation coupled with quantitative mass spectrometry. The mass spectrometry data were extensively analysed using several bioinformatics approaches. RESULTS: The comparative interactome mapping of Tau protein revealed distinct and unique interactors (DPYSL4, ARHGEF2, TUBA4A and UQCRC2) in subtypes of AD. Interestingly, an analysis of the Tau-interacting proteins indicated enrichment of mitochondrial organisation processes, including negative regulation of mitochondrion organisation, mitochondrial outer membrane permeabilisation involved in programmed cell death, regulation of autophagy of mitochondrion and necroptotic processes, specifically in the rpAD interactome. While, in cAD, the top enriched processes were related to oxidation-reduction process, transport and monocarboxylic acid metabolism. CONCLUSIONS: Overall, our results provide a comprehensive map of Tau-interacting protein networks in a subtype-dependent manner and shed light on differential functions/pathways in AD subtypes. This comprehensive map of the Tau-interactome has provided subsets of disease-related proteins that can serve as novel biomarkers/biomarker panels and new drug targets.
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Enfermedad de Alzheimer , Tauopatías , Humanos , Proteínas tau/metabolismo , Enfermedad de Alzheimer/patología , Tauopatías/patología , Encéfalo/patología , Biomarcadores , Factores de Intercambio de Guanina Nucleótido Rho/metabolismoRESUMEN
The microtubule (MT) cytoskeleton and its dynamics play an important role in cell migration. Depletion of the microtubule-severing enzyme Fidgetin-like 2 (FL2), a regulator of MT dynamics at the leading edge of migrating cells, leads to faster and more efficient cell migration. Here we examine how siRNA knockdown of FL2 increases cell motility. Förster resonance energy transfer biosensor studies shows that FL2 knockdown decreases activation of the p21 Rho GTPase, RhoA, and its activator GEF-H1. Immunofluorescence studies reveal that GEF-H1 is sequestered by the increased MT density resulting from FL2 depletion. Activation of the Rho GTPase, Rac1, however, does not change after FL2 knockdown. Furthermore, FL2 depletion leads to an increase in focal adhesion kinase activation at the leading edge, as shown by immunofluorescence studies, but no change in actin dynamics, as shown by fluorescence recovery after photobleaching. We believe these results expand our understanding of the role of MT dynamics in cell migration and offer new insights into RhoA and Rac1 regulation.
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Microtúbulos , Proteína de Unión al GTP rhoA , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Microtúbulos/metabolismo , Movimiento Celular , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein-centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein-coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., "self-activating" behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.
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Amoeba , Naegleria fowleri , Proteínas RGS , Animales , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/metabolismo , Naegleria fowleri/metabolismo , Proteínas RGS/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.
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Factores de Intercambio de Guanina Nucleótido Rho , Proteínas de Unión al GTP rho , Familia-src Quinasas , Fosforilación , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Tirosina/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284-1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.
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Factores de Intercambio de Guanina Nucleótido/química , Proteínas Serina-Treonina Quinasas/química , Factores de Intercambio de Guanina Nucleótido Rho/química , Animales , Microscopía por Crioelectrón , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Neoplasias de la Úvea , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gßγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression-based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein-coupled LPA receptors via the Gßγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gßγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. Taken together, our results indicate that ARHGEF17 may be a valid potential target in the treatment of metastatic lung cancer.