RESUMEN
EXOC5 is a crucial component of a large multi-subunit tethering complex, the exocyst complex, that is required for fusion of secretory vesicles with the plasma membrane. Exoc5 deleted mice die as early embryos. Therefore, to determine the role of EXOC5 in follicular and oocyte development, it was necessary to produce a conditional knockout (cKO), Zp3-Exoc5-cKO, in which Exoc5 was deleted only in oocytes. The first wave of folliculogenesis appeared histologically normal and progressed to the antral stage. However, after IVF with normal sperm, oocytes collected from the first wave (superovulated 21-day-old cKO mice) were shown to be developmentally incompetent. Adult follicular waves did not progress beyond the secondary follicle stage where they underwent apoptosis. Female cKO mice were infertile. Overall, these data suggest that the first wave of folliculogenesis is less sensitive to oocyte-specific loss of Exoc5, but the resulting gametes have reduced developmental competence. In contrast, subsequent waves of folliculogenesis require oocyte-specific Exoc5 for development past the preantral follicle stage. The Zp3-Exoc5-cKO mouse provides a model for disrupting folliculogenesis that also enables the separation between the first and subsequent waves of folliculogenesis.
Asunto(s)
Ratones Noqueados , Oocitos , Oogénesis , Folículo Ovárico , Animales , Femenino , Oogénesis/genética , Oogénesis/fisiología , Oocitos/metabolismo , Ratones , Folículo Ovárico/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Masculino , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismoRESUMEN
Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.
Asunto(s)
Complejo de Reconocimiento del Origen , Cuerpos Polares , Replicación del ADN , Meiosis/genética , Oogénesis/genética , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismoRESUMEN
Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.
Asunto(s)
Diferenciación Celular/genética , Redes Reguladoras de Genes , Nefronas/embriología , Organogénesis/genética , Células Madre/fisiología , Factores de Transcripción/fisiología , Animales , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Proteína Wnt4/genética , Proteína Wnt4/fisiologíaRESUMEN
To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.
Asunto(s)
Células Fotorreceptoras/patología , Degeneración Retiniana , Epitelio Pigmentado de la Retina/patología , Proteínas de Transporte Vesicular/fisiología , Trastornos de la Visión/patología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Trastornos de la Visión/metabolismo , Pez CebraRESUMEN
The recently proposed idea of "urocrine signaling" hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.
Asunto(s)
Cilios/metabolismo , Exocitosis , Vesículas Extracelulares/metabolismo , Riñón/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Células Cultivadas , Perros , Humanos , Células de Riñón Canino Madin Darby/metabolismo , Ratones , Ratones Noqueados , Proteínas de Transporte Vesicular/deficienciaRESUMEN
Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-ß pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.
Asunto(s)
Eliminación de Gen , Glomérulos Renales/patología , Síndrome Nefrótico/patología , Podocitos/patología , Proteínas de Transporte Vesicular/fisiología , Animales , Apoptosis , Movimiento Celular , Exocitosis , Humanos , Glomérulos Renales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome Nefrótico/genética , Fosforilación , Podocitos/metabolismo , Transporte de Proteínas , Proteinuria/etiología , Proteinuria/patología , Transducción de SeñalRESUMEN
BACKGROUND: Bicuspid aortic valve (BAV) disease is a congenital defect that affects 0.5% to 1.2% of the population and is associated with comorbidities including ascending aortic dilation and calcific aortic valve stenosis. To date, although a few causal genes have been identified, the genetic basis for the vast majority of BAV cases remains unknown, likely pointing to complex genetic heterogeneity underlying this phenotype. Identifying genetic pathways versus individual gene variants may provide an avenue for uncovering additional BAV causes and consequent comorbidities. METHODS: We performed genome-wide association Discovery and Replication Studies using cohorts of 2131 patients with BAV and 2728 control patients, respectively, which identified primary cilia genes as associated with the BAV phenotype. Genome-wide association study hits were prioritized based on P value and validated through in vivo loss of function and rescue experiments, 3-dimensional immunohistochemistry, histology, and morphometric analyses during aortic valve morphogenesis and in aged animals in multiple species. Consequences of these genetic perturbations on cilia-dependent pathways were analyzed by Western and immunohistochemistry analyses, and assessment of aortic valve and cardiac function were determined by echocardiography. RESULTS: Genome-wide association study hits revealed an association between BAV and genetic variation in human primary cilia. The most associated single-nucleotide polymorphisms were identified in or near genes that are important in regulating ciliogenesis through the exocyst, a shuttling complex that chaperones cilia cargo to the membrane. Genetic dismantling of the exocyst resulted in impaired ciliogenesis, disrupted ciliogenic signaling and a spectrum of cardiac defects in zebrafish, and aortic valve defects including BAV, valvular stenosis, and valvular calcification in murine models. CONCLUSIONS: These data support the exocyst as required for normal ciliogenesis during aortic valve morphogenesis and implicate disruption of ciliogenesis and its downstream pathways as contributory to BAV and associated comorbidities in humans.
Asunto(s)
Estenosis de la Válvula Aórtica/patología , Válvula Aórtica/anomalías , Cilios/fisiología , Cardiopatías Congénitas/patología , Enfermedades de las Válvulas Cardíacas/patología , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/genética , Enfermedad de la Válvula Aórtica Bicúspide , Estudios de Casos y Controles , Cilios/patología , Frecuencia de los Genes , Estudio de Asociación del Genoma Completo , Genotipo , Cardiopatías Congénitas/genética , Enfermedades de las Válvulas Cardíacas/genética , Enfermedades de las Válvulas Cardíacas/metabolismo , Humanos , Ratones , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Skeletal muscle handles ~80-90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.
Asunto(s)
Exocitosis/genética , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Exocitosis/efectos de los fármacos , Técnicas de Inactivación de Genes , Resistencia a la Insulina , Limoninas/farmacología , Mioblastos Esqueléticos , Transporte de Proteínas/genética , Ratas , Proteínas de Transporte Vesicular/genéticaRESUMEN
We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5, a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins.
Asunto(s)
Cilios/metabolismo , Exocitosis , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Mutación , Células Fotorreceptoras de Vertebrados/patología , Retina/patología , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/metabolismo , Pez CebraRESUMEN
The highly conserved exocyst protein complex regulates polarized exocytosis of subsets of secretory vesicles. A previous study reported that shRNA knockdown of an exocyst central subunit, Sec10 (Sec10-KD) in Madin-Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. We used three-dimensional collagen cultures of MDCK cells to further investigate the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis. Sec10-KD cysts initially demonstrated undisturbed lumen formation although later displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, control cells maintained normal homeostasis, while Sec10-KD cysts displayed numerous apoptotic cells extruded basally into the collagen matrix. Sec10-KD MDCK cells were also more sensitive to apoptotic triggers than controls. These phenotypes were reversed by restoring Sec10 expression with shRNA-resistant human Sec10. Apico-basal polarity appeared normal in Sec10-KD cysts, whereas mitotic spindle angles differed significantly from controls, suggesting a planar cell polarity defect. In addition, analysis of renal tubules in a newly generated kidney-specific Sec10-knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules; abnormal epithelial cell extrusion was also observed, supporting our in vitro results. We hypothesize that, in Sec10-KD cells, the disrupted exocyst activity results in increased apoptotic sensitivity through defective primary cilia signaling and that, in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis.
Asunto(s)
Apoptosis/fisiología , Células Epiteliales/fisiología , Homeostasis/fisiología , Riñón/fisiología , Proteínas de Transporte Vesicular/fisiología , Animales , Proliferación Celular/fisiología , Perros , Humanos , Riñón/citología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.
Asunto(s)
Lesión Renal Aguda/prevención & control , Endocitosis , Receptores ErbB/metabolismo , Túbulos Renales/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Pez Cebra/metabolismo , Lesión Renal Aguda/enzimología , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Perros , Endocitosis/efectos de los fármacos , Activación Enzimática , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Células de Riñón Canino Madin Darby , Estrés Oxidativo , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Factores de Tiempo , Transfección , Proteínas de Transporte Vesicular/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown-including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation-suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.
Asunto(s)
Fenotipo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Canales Catiónicos TRPP/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Cilios/genética , Cilios/metabolismo , Perros , Activación Enzimática/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Riñón/embriología , Riñón/patología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Enfermedades Renales Poliquísticas/patología , Unión Proteica , Canales Catiónicos TRPP/deficiencia , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Transporte Vesicular/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis.
Asunto(s)
Cilios/metabolismo , Células Epiteliales/citología , Células Epiteliales/enzimología , Túbulos Renales/citología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismo , Perros , Activación Enzimática , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
BACKGROUND: Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly made proteins are translocated into or across the RER membrane consists of three main subunits: Sec61α, -ß, and -γ. Sec61ß facilitates translocation, and we and others have shown that the highly conserved eight-protein exocyst complex interacts with Sec61ß. We have also shown that the exocyst is involved in basolateral, not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery has been reported. Furthermore, we have shown that the exocyst is necessary for formation of primary cilia, organelles found on the apical surface. METHODS: GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic endoplasmic reticulum (ER) microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and (35)S-methionine for protein detection were used to investigate the role of the exocyst in synthesis of polarized proteins. P(32)-orthophosphate and immunoprecipitation with antibody against Sec61ß was used to investigate Sec61ß phosphorylation in exocyst Sec10-overexpressing cells. RESULTS: Sec10 biochemically interacts with Sec61ß using GST pulldown. Using cell-free assays, there is enhanced exocyst recruitment to endoplasmic reticulum membranes following exocyst depletion and basolateral G protein of vesicular stomatitis virus protein translation, compared to apical hemagglutinin of influenza virus protein translation. Finally, Sec10 overexpression increases Sec61ß phosphorylation. CONCLUSION: These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61ß.
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Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas/fisiología , Transporte de Proteínas/fisiología , Proteínas de Transporte Vesicular/metabolismo , Animales , Humanos , Conejos , Canales de Translocación SECRESUMEN
Ureter obstruction is a highly prevalent event during embryonic development and is a major cause of pediatric kidney disease. We have previously reported that ureteric bud-specific ablation of the gene expressing the exocyst subunit EXOC5 in late murine gestation results in failure of urothelial stratification, cell death and complete ureter obstruction. However, the mechanistic connection between disrupted exocyst activity, urothelial cell death and subsequent ureter obstruction was unclear. Here, we report that inhibited urothelial stratification does not drive cell death during ureter development. Instead, we demonstrate that the exocyst plays a critical role in autophagy in urothelial cells, and that disruption of autophagy activates a urothelial NF-κB stress response. Impaired autophagy first provokes canonical NF-κB activity, which is progressively followed by increasing levels of non-canonical NF-κB activity and cell death if the stress remains unresolved. Furthermore, we demonstrate that ureter obstructions can be completely rescued in Exoc5 conditional knockout mice by administering a single dose of the pan-caspase inhibitor z-VAD-FMK at embryonic day 16.5 prior to urothelial cell death. Taken together, ablation of Exoc5 disrupts autophagic stress response and activates progressive NF-κB signaling, which promotes obstructive uropathy.
Asunto(s)
Autofagia , FN-kappa B , Animales , Caspasas/metabolismo , Femenino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Embarazo , Transducción de Señal , Proteínas de Transporte Vesicular/genéticaRESUMEN
Cell-cell contacts are essential for epithelial cell function, and disruption is associated with pathological conditions including ischemic kidney injury. We hypothesize that the exocyst, a highly-conserved eight-protein complex that targets secretory vesicles carrying membrane proteins, is involved in maintaining renal epithelial barrier integrity. Accordingly, increasing exocyst expression in renal tubule cells may protect barrier function from oxidative stress resulting from ischemia and reperfusion (I/R) injury. When cultured on plastic, Madin-Darby canine kidney (MDCK) cells overexpressing Sec10, a central exocyst component, formed domes showing increased resistance to hydrogen peroxide (H2O2). Transepithelial electric resistance (TER) of Sec10-overexpressing MDCK cells grown on Transwell filters was higher than in control MDCK cells, and the rate of TER decrease following H2O2 treatment was less in Sec10-overexpressing MDCK cells compared with control MDCK cells. After removal of H2O2, TER returned to normal more rapidly in Sec10-overexpressing compared with control MDCK cells. In collagen culture MDCK cells form cysts, and H2O2 treatment damaged Sec10-overexpressing MDCK cell cysts less than control MDCK cell cysts. The MAPK pathway has been shown to protect animals from I/R injury. Levels of active ERK, the final MAPK pathway step, were higher in Sec10-overexpressing compared with control MDCK cells. U0126 inhibited ERK activation, exacerbated the H2O2-induced decrease in TER and cyst disruption, and delayed recovery of TER following H2O2 removal. Finally, in mice with renal I/R injury, exocyst expression decreased early and returned to normal concomitant with functional recovery, suggesting that the exocyst may be involved in the recovery following I/R injury.
Asunto(s)
Células Epiteliales/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Riñón/enzimología , Sistema de Señalización de MAP Quinasas , Estrés Oxidativo , Daño por Reperfusión/enzimología , Proteínas de Transporte Vesicular/metabolismo , Animales , Butadienos/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Creatinina/sangre , Perros , Impedancia Eléctrica , Activación Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Peróxido de Hidrógeno/farmacología , Riñón/efectos de los fármacos , Riñón/patología , Riñón/fisiopatología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Nitrilos/farmacología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Permeabilidad , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Factores de Tiempo , Transfección , Proteínas de Transporte Vesicular/genéticaRESUMEN
The original article contains an error for a grant number in the Acknowledgements section.
RESUMEN
One requirement for establishing polarity within a cell is the asymmetric trafficking of intracellular vesicles to the plasma membrane. This tightly regulated process creates spatial and temporal differences in both plasma membrane composition and the membrane-associated proteome. Asymmetric membrane trafficking is also a critical mechanism to regulate cell differentiation, signaling, and physiology. Many eukaryotic cell types use the eight-protein exocyst complex to orchestrate polarized vesicle trafficking to certain membrane locales. Members of the exocyst were originally discovered in yeast while screening for proteins required for the delivery of secretory vesicles to the budding daughter cell. The same eight exocyst genes are conserved in mammals, in which the specifics of exocyst-mediated trafficking are highly cell-type-dependent. Some exocyst members bind to certain Rab GTPases on intracellular vesicles, whereas others localize to the plasma membrane at the site of exocytosis. Assembly of the exocyst holocomplex is responsible for tethering these vesicles to the plasma membrane before their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis. In this review, we will focus on the role and regulation of the exocyst complex in targeted vesicular trafficking as related to the establishment and maintenance of cellular polarity. We will contrast exocyst function in apicobasal epithelial polarity versus front-back mesenchymal polarity, and the dynamic regulation of exocyst-mediated trafficking during cell phenotype transitions.
Asunto(s)
Polaridad Celular , Exocitosis , Animales , Transporte Biológico , Movimiento Celular , Citoesqueleto/fisiología , Transición Epitelial-Mesenquimal , Humanos , Uniones Intercelulares/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Vesículas Secretoras/fisiologíaRESUMEN
The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1Cre mice with Exoc5f/f mice for hair cell-specific deletion (Gfi1Cre/+;Exoc5f/f) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5f/f). Gfi1Cre/+;Exoc5f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.
Asunto(s)
Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Audición , Neuronas/patología , Ganglio Espiral de la Cóclea/patología , Proteínas de Transporte Vesicular/metabolismo , Animales , Apoptosis , Supervivencia Celular , Proteínas de Unión al ADN/metabolismo , Dependovirus/metabolismo , Epitelio/patología , Células Ciliadas Auditivas/ultraestructura , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Integrasas/metabolismo , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Neuritas/metabolismo , Neuronas/metabolismo , Órgano Espiral/metabolismo , Órgano Espiral/ultraestructura , Estereocilios/metabolismo , Estereocilios/ultraestructura , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/deficienciaRESUMEN
We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with beta-aminopropionitrile (betaAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of betaAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with betaAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on betaAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer.