RESUMEN
Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90 % mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response.
Asunto(s)
Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Filogenia , Ranavirus , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Inmunidad Innata/genética , Lubina/inmunología , Ranavirus/fisiología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Regulación de la Expresión Génica/inmunología , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos , Perfilación de la Expresión Génica/veterinariaRESUMEN
The exocyst complex is an important regulator of intracellular trafficking and tethers secretory vesicles to the plasma membrane. Understanding of its role in neuron outgrowth remains incomplete, and previous studies have come to different conclusions about its importance for axon and dendrite growth, particularly in vivo. To investigate exocyst function in vivo we used Drosophila sensory neurons as a model system. To bypass early developmental requirements in other cell types, we used neuron-specific RNAi to target seven exocyst subunits. Initial neuronal development proceeded normally in these backgrounds, however, we considered this could be due to residual exocyst function. To probe neuronal growth capacity at later times after RNAi initiation, we used laser microsurgery to remove axons or dendrites and prompt regrowth. Exocyst subunit RNAi reduced axon regeneration, although new axons could be specified. In control neurons, a vesicle trafficking marker often concentrated in the new axon, but this pattern was disrupted in Sec6 RNAi neurons. Dendrite regeneration was also severely reduced by exocyst RNAi, even though the trafficking marker did not accumulate in a strongly polarized manner during normal dendrite regeneration. The requirement for the exocyst was not limited to injury contexts as exocyst subunit RNAi eliminated dendrite regrowth after developmental pruning. We conclude that the exocyst is required for injury-induced and developmental neurite outgrowth, but that residual protein function can easily mask this requirement.
Asunto(s)
Axones , Exocitosis , Exocitosis/fisiología , Neuritas , Regeneración Nerviosa , Membrana Celular/metabolismoRESUMEN
Exocytosis is an active vesicle trafficking process by which eukaryotes secrete materials to the extracellular environment and insert membrane proteins into the plasma membrane. The final step of exocytosis in yeast involves the assembly of two t-SNAREs, Sso1/2 and Sec9, with the v-SNARE, Snc1/2, on secretory vesicles. The rate-limiting step in this process is the formation of a binary complex of the two t-SNAREs. Despite a previous report of acceleration of binary complex assembly by Sec3, it remains unknown how Sso2 is efficiently recruited to the vesicle-docking site marked by Sec3. Here, we report a crystal structure of the pleckstrin homology (PH) domain of Sec3 in complex with a nearly full-length version of Sso2 lacking only its C-terminal transmembrane helix. The structure shows a previously uncharacterized binding site for Sec3 at the N-terminus of Sso2, consisting of two highly conserved triple residue motifs (NPY: Asn-Pro-Tyr). We further reveal that the two NPY motifs bind Sec3 synergistically, which together with the previously reported binding interface constitute dual-site interactions between Sso2 and Sec3 to drive the fusion of secretory vesicles at target sites on the plasma membrane.
Asunto(s)
Proteínas SNARE , Proteínas de Saccharomyces cerevisiae , Membrana Celular/metabolismo , Exocitosis , Fusión de Membrana , Proteínas Qa-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Neurons are the largest known cells, with complex and highly polarized morphologies and consist of a cell body (soma), several dendrites, and a single axon. The establishment of polarity necessitates initial axonal outgrowth in concomitance with the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles primarily at the neuronal growth cone membrane. The multiprotein exocyst complex drives spatial location and specificity of vesicle fusion at plasma membrane. However, the specific participation of its different proteins on neuronal differentiation has not been fully established. In the present work we analyzed the role of Sec3, a prominent exocyst complex protein on neuronal differentiation. Using mice hippocampal primary cultures, we determined that Sec3 is expressed in neurons at early stages prior to neuronal polarization. Furthermore, we determined that silencing of Sec3 in mice hippocampal neurons in culture precluded polarization. Moreover, using in utero electroporation experiments, we determined that Sec3 knockdown affected cortical neurons migration and morphology during neocortex formation. Our results demonstrate that the exocyst complex protein Sec3 plays an important role in axon formation in neuronal differentiation and the migration of neuronal progenitors during cortex development.
Asunto(s)
Corteza Cerebral/embriología , Neurogénesis/fisiología , Neuronas , Proteínas de Transporte Vesicular/metabolismo , Animales , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Corteza Cerebral/metabolismo , Ratones , Neuronas/citología , Neuronas/metabolismoRESUMEN
The exocyst, an evolutionarily conserved octomeric protein complex, has been demonstrated as an essential component for vesicle tethering during cell exocytosis, and participates in various physiological processes in the cell. Although subunits of the exocyst complex have been reported to be involved in the regulation of TGF-ß induced cancer cell migration and epithelial-mesenchymal transition (EMT), the potential function of Sec3 in these regulated processes remains unclear. Here, we show that Sec3 knockdown abolishes TGF-ß stimulated A549 lung cancer cell migration in vitro and causes defects in the regulated EMT process. In addition, we find that depletion of Sec3 significantly inhibits TGF-ß stimulated Akt phosphorylation in A549 cells, whereas the increase of Smad2 phosphorylation is unaffected. Furthermore, replenishment of an RNAi-resistant form of Sec3 is shown to restore the defects of TGF-ß induced cell migration, EMT and Akt signaling activation. In summary, our study provides evidence that Sec3 is involved in TGF-ß induced cell migration and EMT processes, presumably through the regulation of PI3K/Akt signaling activation in A549 cancer cells.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/farmacología , Proteínas de Transporte Vesicular/deficiencia , Células A549 , Movimiento Celular/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Cell polarity refers to spatial difference in morphology, structure, and function within different parts of a single cell, which plays important roles in a wide range of cellular processes. In eukaryotic cells, the small GTPase Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) are critical components for cell polarity and required for polarized exocytosis and cell growth. Previous data showed that the GTPase-interacting components, Gic1 and Gic2, control cell polarity through its binding with Cdc42 and PtdIns(4,5)P2 in the plasma membrane in budding yeast. However, whether the Gic proteins regulate polarized exocytosis is unknown. RESULTS: In this study, we found that Gic2 co-immunoprecipitates with the exocyst complex, suggesting Gic proteins may be involved in exocytosis. Although we could not show the direct interaction between Gic2 and exocyst, we found gic1Δgic2Δ are synthetically sick with sec3ΔN. We demonstrated that Gic1 and Gic2 are required for polarized exocytosis in a yeast strain harboring the N-terminal domain deletion of Sec3, which is also known as an effector of Cdc42 GTPase. Gic proteins are required for polarized localization of exocyst, growth, and efficient secretion in sec3∆N mutant. In addition, we found that the N-terminal domain of both Gic2 and Sec3 share the similar binding sites of Cdc42. Surprisingly, not all the Sec3/Gic binding deficient cdc42 mutants displayed defects of growth and secretion, indicating that disruption of Cdc42 binding with Gic proteins and Sec3 does not necessarily show secretion defects in cdc42 mutants. CONCLUSIONS: We conclude that Gic1/2 and Sec3 act in parallel to regulate polarized post-Golgi secretion, but this regulation is not solely controlled by their upstream factor Cdc42. Considering that N-terminal domain of Gic2 and Sec3 can bind to both Cdc42 and PtdIns(4,5)P2, the regulation of Gic protein and Sec3 on polarized secretion may also be controlled by PtdIns(4,5)P2. Further experiments need to be performed to test this hypothesis. Our findings provide important clues for understanding the molecular mechanism of cell polarity establishment in eukaryotic cells.
RESUMEN
The exocyst complex plays multiple roles via tethering secretory or recycling vesicles to the plasma membrane. Previous studies have demonstrated that the exocyst contains eight components, which possibly have some redundant but distinct functions. It is therefore interesting to investigate the biological function of each component. Here, we found that Sec3, one component of exocyst complex, is involved in Drosophila egg chamber development. Loss of sec3 results in egg chamber fusion through the abolishment of cell differentiation. In addition, loss of sec3 increases cell numbers but decreases cell size. These defects phenocopy Notch pathway inactivation. In line with this, loss of sec3 indeed leads to Notch protein accumulation, suggesting that the loss of Sec3 inhibits the delivery of Notch onto the plasma membrane and accumulates inactive Notch in the cytoplasm. Loss of sec3 also leads to the ectopic expression of two Notch pathway target genes, Cut and FasciclinIII, which should normally be downregulated by Notch. Altogether, our study revealed that Sec3 governs egg chamber development through the regulation of Notch, and provides fresh insights into the regulation of oogenesis.
RESUMEN
NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.
Asunto(s)
Proteínas de Ciclo Celular/genética , Exocitosis/genética , Regulación Fúngica de la Expresión Génica , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Citocinesis/genética , Fosfoproteínas/clasificación , Fosfoproteínas/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
AIM: To construct a lentiviral vector with endostatin (ES) and staphylococcal enterotoxin C3(SEC3) gene, and investigate its capacities of inhibition on proliferation and migration of Hela cells. METHODS: By inserting ES and SEC3 gene into the plasmid and then transfect 293 T cell, the co-expressed (SEC3-ES) vector were constructed. A series of experiments in vitro were carried out to detect its anti-tumor capacity. RESULTS: SEC3 expression of the vector is about 3 times of GV365-SEC3 vector, and ES expression is over 22.5-fold compared with GV365-ES vector. Moreover, OD490 value of CO group (1.212 ± 0.003) was notably lower than NC (negative control) group (1.124 ± 0.01) (P < 0.05) in MTT assay. Cell cycle analysis showed it could block Hela cells in S phase. Meanwhile, in wound healing assay, cells of CO group migrated at a slower rate (0.59 ± 0.02) compared with NC group (0.65 ± 0.02)(P < 0.01). CONCLUSION: The successful construction of co-expressed vector lays the foundation for further studies in vivo. These promising results suggest a new strategy to treating cervical cancer.
Asunto(s)
Antineoplásicos/farmacología , Endostatinas/genética , Enterotoxinas/genética , Terapia Genética/métodos , Vectores Genéticos , Movimiento Celular , Proliferación Celular , Femenino , Células HEK293 , Células HeLa , Humanos , Lentivirus , Transfección , Neoplasias del Cuello Uterino/virologíaRESUMEN
Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and tightly regulated by a variety of factors. The exocyst complex is one of the multi-subunit tethering complexes and functions in the tethering of the secretory vesicles to the plasma membrane. We have found that the yeast Sec3, a subunit of the exocyst, binds to the t-SNARE protein Sso2 and promotes its interaction with another t-SNARE protein, Sec9. Here, we describe the structural analysis and in vitro membrane fusion assays, by which we found that Sec3 binding leads to a conformational change within Sso2, and facilitates SNARE assembly and the membrane fusion.
Asunto(s)
Fusión de Membrana , Modelos Moleculares , Proteínas Qa-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos/genética , Liposomas/química , Liposomas/metabolismo , Mutagénesis , Unión Proteica , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/aislamiento & purificación , Proteínas Qc-SNARE/química , Proteínas Qc-SNARE/aislamiento & purificación , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.
Asunto(s)
Membrana Celular/química , Exocitosis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Membrana Celular/metabolismo , Cinética , Simulación de Dinámica Molecular , Mutación , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vías Secretoras , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
During collective migration, guidance receptors signal downstream to result in a polarized distribution of molecules, including cytoskeletal regulators and guidance receptors themselves, in response to an extracellular gradient of chemotactic factors. However, the underlying mechanism of asymmetry generation in the context of the migration of a group of cells is not well understood. Using border cells in the Drosophila ovary as a model system for collective migration, we found that the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) is required for a polarized distribution of recycling endosome and exocyst in the leading cells of the border cell cluster. Interestingly, PVR signaled through the small GTPase Rac to positively affect the levels of Rab11-labeled recycling endosomes, probably in an F-actin-dependent manner. Conversely, the exocyst complex component Sec3 was required for the asymmetric localization of RTK activity and F-actin, similar to that previously reported for the function of Rab11. Together, these results suggested a positive-feedback loop in border cells, in which RTKs such as PVR act to induce a higher level of vesicle recycling and tethering activity in the leading cells, which in turn enables RTK activity to be distributed in a more polarized fashion at the front. We also provided evidence that E-cadherin, the major adhesion molecule for border cell migration, is a specific cargo in the Rab11-labeled recycling endosomes and that Sec3 is required for the delivery of the E-cadherin-containing vesicles to the membrane.