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Trigonella, commonly known as Fenugreek, is among the most promising medicinal herbs consumed worldwide due its protein rich dietary contributions. This study involved induced mutagenesis on two Trigonella species (Trigonella foenum-graecum var. PEB and Trigonella corniculata var. Pusa kasuri) using caffeine and sodium azide as mutagens, resulting in the identification of nine high-yielding mutant lines in the M3 generation. Molecular characterization using SCoT markers revealed a high polymorphism of 28.3% and 46.7% in PEB and Pusa kasuri, respectively, facilitating the investigation of genetic divergence among the control and mutant lines. Similarity correlation analysis indicated a high similarity between mutant A and mutant C (0.97) and between mutant J and mutant O (0.88), while the lowest similarity was observed between mutant B and mutant F (0.74) and between control and mutant L (0.58). Mutant F and Mutant J displayed the highest seed yield and its attributing traits, and seed protein content in PEB and Pusa kasuri, respectively. Physiological parameters, including chlorophyll content (Mutants A and N) and carotenoids (mutant A and J), exhibited improvements. Assessment of stomatal and seed characteristics using scanning electron microscopy may lead to improved physiological processes and distinction at the interspecific level, respectively. Methanolic extracts of the control and the mutant lines of both species were subjected to GC-MS analysis, revealing 24 major phytocompounds known for their pharmacological activities (antioxidant, anti-inflammatory, anticancer, etc.). Statistical methods such as Pearson correlation heatmap and pairwise scatter plot matrix provided insights into the correlations and linear associations among parameters for both PEB and Pusa kasuri. The strong correlation between iron content and seeds per pod in the mutant lines suggests a promising avenue for further research. Continued research and breeding efforts using these mutants can lead to significant advancements in agriculture and medicine, benefiting farmers, consumers, and industries alike.
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Mutación , Semillas , Trigonella , Trigonella/genética , Trigonella/química , Trigonella/metabolismo , Semillas/genética , Semillas/metabolismo , Semillas/química , Mutagénesis , Carotenoides/metabolismo , Cafeína/farmacología , Clorofila/metabolismo , Azida Sódica/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Plant breeding, aimed at enhancing desired traits, depends on genetic diversity. Mutation breeding is a powerful method of rapidly expanding genetic diversity, facilitating crop improvement, and ensuring food security. In a recent study, researchers evaluated the genetic variability of Trigonella species using different doses of sodium azide (SA) (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) through morphological, physiological, and cytogenetic studies. Morphological variations were observed in cotyledonary leaves, vegetative leaves, and overall plant growth and habit. Several quantitative parameters, such as plant height, fertile branches per plant, pods per plant (or clusters), seeds per pod, and seed yield, increased when treated with 0.2% and 0.4% SA compared to the control. Furthermore, the total chlorophyll content and carotenoids increased in the sample treated with 0.2% SA over the control but decreased with higher concentrations. Scanning electron microscopy revealed that stomatal aperture and seed dimensions increased at lower concentrations of sodium azide treatment. The study found a positive correlation between the different parameters studied in the Trigonella species, as indicated by high r-values. Based on their findings, it was concluded that the genotype of fenugreek can be improved by using 0.2% and 0.4% concentrations of sodium azide. However, the evaluation of observed variants in successive generations is a critical and necessary process to validate their potential as keystones for crop genetic improvements.
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Daylight vision is mediated by cone photoreceptors in vertebrates, which synapse with bipolar cells (BCs) and horizontal (HCs) cells. This cone synapse is functionally and anatomically complex, connecting to 8 types of depolarizing BCs (DBCs) and 5 types of hyperpolarizing BCs (HBCs) in mice. The dendrites of DBCs and HCs cells make invaginating ribbon synapses with the cone axon terminal, while HBCs form flat synapses with the cone pedicles. The molecular architecture that underpins this organization is relatively poorly understood. To identify new proteins involved in synapse formation and function we used an unbiased proteomic approach and identified LRFN2 (leucine-rich repeat and fibronectin III domain-containing 2) as a component of the DBC signaling complex. LRFN2 is selectively expressed at cone terminals and co-localizes with PNA, and other DBC signalplex members. In LRFN2 deficient mice, the synaptic markers: LRIT3, ELFN2, mGluR6, TRPM1 and GPR179 are properly localized. Similarly, LRFN2 expression and localization is not dependent on these synaptic proteins. In the absence of LRFN2 the cone-mediated photopic electroretinogram b-wave amplitude is reduced at the brightest flash intensities. These data demonstrate that LRFN2 absence compromises normal synaptic transmission between cones and cone DBCs.Significance Statement Signaling between cone photoreceptors and the downstream bipolar cells is critical to normal vision. Cones synapse with 13 different types of bipolar cells forming an invaginating ribbon synapses with 8 types, and flat synapses with 5 types, to form one of the most complex synapses in the brain. In this report a new protein, LRFN2 (leucine-rich repeat and fibronectin III domain-containing 2), was identified that is expressed at the cone synapse. Using Lrfn2 knockout mice we show LRFN2 is required for the normal cone signaling.
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Complete congenital stationary night blindness (cCSNB) is a heterogeneous disorder characterized by poor dim-light vision, myopia, and nystagmus that is caused by mutations in genes critical for signal transmission between photoreceptors and depolarizing bipolar cells (DBCs). One such gene, LRIT3, is required for assembly of the post-synaptic signaling complex (signalplex) at the dendritic tips of DBCs, although the number of signalplex components impacted is greater in cone DBCs (all components) than in rod bipolar cells (only TRPM1 and Nyctalopin). Here we show that rAAV-mediated expression of LRIT3 in cones results in robust rescue of cone DBC signalplex components and partially restores downstream visual function, as measured by the light-adapted electroretinogram (ERG) b-wave and electrophysiological recordings of bipolar cells (BCs) and RGCs. These data show that LRIT3 successfully restores partial function to cone DBCs most likely in a trans-synaptic manner, potentially paving the way for therapeutic intervention in LRIT3-associated cCSNB.
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Trigonella foenum graecum L. (Fenugreek) is a valuable medicinal plant cultivated for decades for its therapeutic characteristics. Still no pronounced improvement concerning wild form was accomplished as it is a self-pollinating crop. Induced mutagenesis is encouraged as a remarkable tool on this plant to circumvent the genetic bottleneck of cultivated germplasms. As a result, novel allelomorphic combinations for short-term agronomic attributes were developed. Fenugreek cultivar Pusa Early Bunching, selected for the present experiment, was mutagenized with five doses (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) of caffeine and sodium azide (SA) to evaluate its impact on the qualitative and quantitative traits of M1 and M2 generation conducted in a Complete Randomized Block Design (CRBD), replicated five times during 2019-2020 and 2020-2021, respectively. The frequency of induced phenotypic variations was assessed in M2 progenies, resulting in the identification and isolation of a broad spectrum of mutants with altered phenotypes. Mutagenic effectiveness and efficiency were found to be maximum at lower concentrations of the mutagen treatments and highest in SA, followed by caffeine. Various morphological mutants with modified characters were observed at different concentrations in M2 generation. The spectrum of mutations was wider in SA than in caffeine, as caffeine produced 51 while SA produced 54 individual mutants under seven major categories. The maximum frequency of morphological mutants was associated with leaf, followed by plant size, plant growth habit, pod, seed size, seed shape, and seed color. Morphological and structural variations in the guard cells of stomata and seeds were observed through scanning electron microscopy. The variations created in the economically important traits may enrich the genetic diversity of this plant species. Moreover, these morphological mutants may serve as a source of elite genes in further breeding programs of fenugreek.
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BACKGROUND: DNA markers improved the productivity and accuracy of classical plant breeding by means of marker-assisted selection (MAS). The enormous number of quantitative trait loci (QTLs) mapping read for different plant species have given a plenitude of molecular marker-gene associations. In this review, we have discussed the positive aspects of molecular marker-assisted selection and its precise applications in plant breeding programmes. Molecular marker-assisted selection has considerably shortened the time for new crop varieties to be brought to the market. To explore the information about DNA markers, many reviews have been published in the last few decades; all these reviews were intended by plant breeders to obtain information on molecular genetics. In this review, we intended to be a synopsis of recent developments of DNA markers and their application in plant breeding programmes and devoted to early breeders with little or no knowledge about the DNA markers. The progress made in molecular plant breeding, plant genetics, genomics selection, and editing of genome contributed to the comprehensive understanding of DNA markers and provides several proofs on the genetic diversity available in crop plants and greatly complemented plant breeding devices. SHORT CONCLUSION: MAS has revolutionized the process of plant breeding with acceleration and accuracy, which is continuously empowering plant breeders around the world.
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The first retinal synapse, photoreceptorâbipolar cell (BC), is both anatomically and functionally complex. Within the same synaptic region, a change in presynaptic glutamate release is sensed by both ON BCs (DBCs) via the metabotropic glutamate receptor 6 (mGluR6), and OFF BCs (HBCs) via ionotropic glutamate receptors to establish parallel signaling pathways that preferentially encode light increments (ON) or decrements (OFF), respectively. The synaptic structural organization of ON and OFF-type BCs at the photoreceptor terminal differs. DBCs make an invaginating synapse that contains a diverse but incompletely understood complex of interacting proteins (signalplex). HBCs make primarily flat contacts that contain an apparent different set of proteins that is equally uncharacterized. LRIT3 is a synaptic protein known to be essential for ON pathway visual function. In both male and female mice, we demonstrate that LRIT3 interacts with and is required for expression of nyctalopin, and thus TRPM1 at all DBC dendritic tips, but DBC signalplex components are not required for LRIT3 expression. Using whole-cell and multielectrode array (MEA) electrophysiology and glutamate imaging, we demonstrate that the loss of LRIT3 impacts both ON and OFF signaling pathway function. Without LRIT3, excitatory input to type 1 BCs is reduced, as are the visually evoked responses of many OFF retinal ganglion cells (RGCs). We conclude that the absence of LRIT3 expression disrupts excitatory input to OFF BCs and, thus disrupts the normal function of OFF RGCs.
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Proteínas de la Membrana , Retina , Células Bipolares de la Retina , Transducción de Señal , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , SinapsisRESUMEN
Breast cancer is the second leading cause of cancer-related deaths among women and 90% of these mortalities can be attributed to progression to metastatic disease. In particular, triple negative breast cancer (TNBC) is extremely aggressive and frequently metastasizes to multiple organs. As TNBCs are categorized by their lack of hormone receptors, these tumors are very heterogeneous and are immune to most targeted therapies. Metabolic changes are observed in the majority of TNBC and a large proportion upregulate enzymes within the serine synthesis pathway, including phosphoserine aminotransferase 1 (PSAT1). In this report, we investigate the role of PSAT1 in migration and invasion potential in a subset of TNBC cell types. We found that the expression of PSAT1 increases with TNBC clinical grade. We also demonstrate that suppression of PSAT1 or phosphoglycerate dehydrogenase (PHGDH) does not negatively impact cell proliferation in TNBC cells that are not dependent on de novo serine synthesis. However, we observed that suppression of PSAT1 specifically alters the F-actin cytoskeletal arrangement and morphology in these TNBC cell lines. In addition, suppression of PSAT1 inhibits motility and migration in these TNBC cell lines, which is not recapitulated upon loss of PHGDH. PSAT1 silencing also reduced the number of lung tumor nodules in a model of experimental metastasis; yet did not decrease anchorage-independent growth. Together, these results suggest that PSAT1 functions to drive migratory potential in promoting metastasis in select TNBC cells independent of its role in serine synthesis.
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Mama/patología , Neoplasias Pulmonares/secundario , Pulmón/patología , Transaminasas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Mama/cirugía , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Clasificación del Tumor , Invasividad Neoplásica/genética , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , ARN Interferente Pequeño/metabolismo , Análisis de Matrices Tisulares , Transaminasas/análisis , Transaminasas/genética , Neoplasias de la Mama Triple Negativas/cirugía , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Throughout the CNS, interactions between pre- and postsynaptic adhesion molecules establish normal synaptic structure and function. Leucine-rich repeat (LRR) domain-containing proteins are a large family that has a diversity of ligands, and their absence can cause disease. At the first retinal synapse, the absence of LRIT3 expression leads to the disassembly of the postsynaptic glutamate signaling complex (signalplex) expressed on depolarizing bipolar cell (DBC) dendrites. The prevalent view is that assembly of the signalplex results from direct postsynaptic protein:protein interactions. In contrast, we demonstrate that LRIT3 is expressed presynaptically, in rod photoreceptors (rods), and when we restore LRIT3 expression in Lrit3-/- rods, we restore expression of the postsynaptic glutamate signalplex and rod-driven vision. Our results demonstrate that, in the retina, the LRR-containing protein LRIT3 acts as a transsynaptic organizer of the postsynaptic complex required for normal synaptic function.
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Ácido Glutámico/metabolismo , Proteínas de la Membrana/metabolismo , Sinapsis/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Dendritas/metabolismo , Dendritas/fisiología , Femenino , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Células Bipolares de la Retina/metabolismo , Células Bipolares de la Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/fisiología , Sinapsis/fisiología , Potenciales SinápticosRESUMEN
Purpose: The Grm6nob8 mouse carries a missense mutation in the Grm6 gene (p.Met66Leu), and exhibits a reduced b-wave of the electroretinogram (ERG), abnormal localization of metabotropic glutamate receptor 6 (mGluR6) to the depolarizing bipolar cell (DBC) soma, and a reduced level of mGluR6 at the DBC dendritic tips. Although the underlying mechanism remains unknown, one possible explanation is that DBCs cannot efficiently traffic the mutant mGluR6. In that scenario, reducing the total amount of mutant mGluR6 protein might normalize localization, and thus, improve the ERG phenotype as well. The second purpose of this study was to determine whether the abnormal cellular distribution of mutant mGluR6 in Grm6nob8 retinas might induce late onset DBC degeneration. Methods: We crossed Grm6nob8 animals with Grm6nob3 mice, which carry a null mutation in Grm6, to generate Grm6nob3/nob8 compound heterozygotes. We used western blotting to measure the total mGluR6 content, and immunohistochemistry to document mGluR6 localization within DBCs. In addition, we examined outer retinal function with ERG and retinal architecture in vivo with spectral domain optical coherence tomography (SD-OCT). Results: The retinal content of mGluR6 was reduced in the retinas of the Grm6nob3/nob8 compound heterozygotes compared to the Grm6nob8 homozygotes. The cellular distribution of mGluR6 in the Grm6nob3/nob8 compound heterozygotes matched that of the Grm6nob8 homozygotes, with extensive expression throughout the DBC cell body and limited expression at the DBC dendritic tips. The dark-adapted ERG b-waves of the Grm6nob3/nob8 mice were reduced in comparison to those of the Grm6nob8 homozygotes at postnatal day 21 and 28. The overall ERG waveforms obtained from 4- through 68-week old Grm6nob8 mice were in general agreement for dark- and light-adapted conditions. The maximum response and sensitivity of the dark-adapted ERG b-wave did not change statistically significantly with age. SD-OCT revealed the maintained laminar structure of the retina, including a clear inner nuclear layer (INL) at each age examined (from 11 to 57 weeks old), although the INL in the mice older than 39 weeks of age was somewhat thinner than that seen at 11 weeks. Conclusions: Mislocalization of mutant mGluR6 is not normalized by reducing the total mGluR6. Mislocalized mutant mGluR6 does not trigger substantial loss of DBCs.
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Regulación de la Expresión Génica , Receptores de Glutamato Metabotrópico/metabolismo , Retina/metabolismo , Animales , Adaptación a la Oscuridad , Electrorretinografía , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Fenotipo , Proteína Quinasa C-alfa/metabolismo , Tomografía de Coherencia ÓpticaRESUMEN
GRM6 encodes the metabotropic glutamate receptor 6 (mGluR6) used by retinal depolarizing bipolar cells (DBCs). Mutations in GRM6 lead to DBC dysfunction and underlie the human condition autosomal recessive complete congenital stationary night blindness. Mouse mutants for Grm6 are important models for this condition. Here we report a new Grm6 mutant, identified in an electroretinogram (ERG) screen of mice maintained at The Jackson Laboratory. The Grm6nob8 mouse has a reduced-amplitude b-wave component of the ERG, which reflects light-evoked DBC activity. Sequencing identified a missense mutation that converts a highly conserved methionine within the ligand binding domain to leucine (p.Met66Leu). Consistent with prior studies of Grm6 mutant mice, the laminar size and structure in the Grm6nob8 retina were comparable to control. The Grm6nob8 phenotype is distinguished from other Grm6 mutants that carry a null allele by a reduced but not absent ERG b-wave, decreased but present expression of mGluR6 at DBC dendritic tips, and mislocalization of mGluR6 to DBC somas. Consistent with a reduced but not absent b-wave, there were a subset of retinal ganglion cells whose responses to light onset have times to peak within the range of those in control retinas. These data indicate that the p.Met66Leu mutant mGluR6 is trafficked less than control. However, the mGluR6 that is localized to the DBC dendritic tips is able to initiate DBC signal transduction. The Grm6nob8 mouse extends the Grm6 allelic series and will be useful for elucidating the role of mGluR6 in DBC signal transduction and in human disease.NEW & NOTEWORTHY This article describes a mouse model of the human disease complete congenital stationary night blindness in which the mutation reduces but does not eliminate GRM6 expression and bipolar cell function, a distinct phenotype from that seen in other Grm6 mouse models.
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Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Mutación Missense , Miopía/metabolismo , Ceguera Nocturna/metabolismo , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Células Bipolares de la Retina/metabolismo , Visión Ocular/fisiología , Animales , Dendritas/metabolismo , Dendritas/patología , Dendritas/efectos de la radiación , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas de Escherichia coli , Enfermedades Hereditarias del Ojo/genética , Enfermedades Hereditarias del Ojo/patología , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Miopía/genética , Miopía/patología , Ceguera Nocturna/genética , Ceguera Nocturna/patología , Células Bipolares de la Retina/patología , Factores de TranscripciónRESUMEN
Cacna1s encodes the α1S subunit (Cav1.1) of voltage-dependent calcium channels, and is required for normal skeletal and cardiac muscle function, where it couples with the ryanodine receptor to regulate muscle contraction. Recently CACNA1S was reported to be expressed on the tips of retinal depolarizing bipolar cells (DBCs) and colocalized with metabotropic glutamate receptor 6 (mGluR6), which is critical to DBC signal transduction. Further, in mGluR6 knockout mice, expression at this location is down regulated. We examined RNAseq data from mouse retina and found expression of a novel isoform of Cacna1s. To determine if CACNA1S was a functional component of the DBC signal transduction cascade, we performed immunohistochemistry to visualize its expression in several mouse lines that lack DBC function. Immunohistochemical staining with antibodies to CACNA1S show punctate labeling at the tips of DBCs in wild type (WT) retinas that are absent in Gpr179 nob5 mutant retinas and decreased in Grm6 -/- mouse retinas. CACNA1S and transient receptor potential cation channel, subfamily M, member 1 (TRPM1) staining also colocalized in WT retinas. Western blot analyses for CACNA1S of either retinal lysates or proteins after immunoprecipitation with the CACNA1S antibody failed to show the presence of bands expected for CACNA1S. Mass spectrometric analysis of CACNA1S immunoprecipitated proteins also failed to detect any peptides matching CACNA1S. Immunohistochemistry and western blotting after expression of GPR179 in HEK293T cells indicate that the CACNA1S antibody used here and in the retinal studies published to date, cross-reacts with GPR179. These data suggest caution should be exercised in conferring a role for CACNA1S in DBC signal transduction based solely on immunohistochemical staining.
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Reacciones Antígeno-Anticuerpo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/inmunología , Regulación de la Expresión Génica/fisiología , Receptores Acoplados a Proteínas G/inmunología , Retina/metabolismo , Células Bipolares de la Retina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Reacciones Cruzadas , Femenino , Células HEK293 , Humanos , Inmunohistoquímica , Síndromes de Inmunodeficiencia , Masculino , Espectrometría de Masas , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Enfermedades de Inmunodeficiencia Primaria , Isoformas de Proteínas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
Parallel visual pathways are initiated at the first retinal synapse by signaling between the rod and cone photoreceptors and two general classes of bipolar cells. For normal function, ON or depolarizing bipolar cells (DBCs) require the G-protein-coupled receptor, mGluR6, an intact G-protein-coupled cascade and the transient receptor potential melastatin 1 (TRPM1) cation channel. In addition, another seven transmembrane protein, GPR179, is required for DBC function and recruits the regulators of G-protein signaling (RGS) proteins, RGS7 and RGS11, to the dendritic tips of the DBCs. Here we use the Gpr179(nob5) mouse, which lacks GPR179 and has a no b-wave electroretinogram (ERG) phenotype, to demonstrate that despite the absence of both GPR179 and RGS7/RGS11, a small dark-adapted ERG b-wave remains and can be enhanced with long duration flashes. Consistent with the ERG, the mGluR6-mediated gating of TRPM1 can be evoked pharmacologically in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs if strong stimulation conditions are used. In contrast, direct gating of TRPM1 by capsaicin in RGS7(-/-)/RGS11(-/-) and WT rod BCs is similar, but severely compromised in Gpr179(nob5) rod BCs. Noise and standing current analyses indicate that the remaining channels in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs have a very low open probability. We propose that GPR179 along with RGS7 and RGS11 controls the ability of the mGluR6 cascade to gate TRPM1. In addition to its role in localizing RGS7 and RGS11 to the dendritic tips, GPR179 via a direct interaction with the TRPM1 channel alters its ability to be gated directly by capsaicin.
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Regulación de la Expresión Génica/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Células Bipolares de la Retina/metabolismo , Transducción de Señal/fisiología , Animales , Capsaicina/farmacología , Línea Celular Transformada , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica/genética , Glicina/análogos & derivados , Glicina/farmacología , Glicinérgicos/farmacología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteoglicanos/metabolismo , Receptores de GABA-A/genética , Retina/citología , Retina/metabolismo , Células Bipolares de la Retina/citología , Células Bipolares de la Retina/efectos de los fármacos , Transducción de Señal/genética , Estricnina/farmacología , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion(1-4). Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion(5). In a cell fusion assay(6,7), SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively. Here we describe a new assay(8) that quantifies SNARE-mediated cell fusion events by activated expression of ß-galactosidase. Two components of the Tet-Off gene expression system(9) are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of ß-galactosidase. The activity of ß-galactosidase is quantified using a colorimetric method by absorbance at 420 nm. The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments(10-15). By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.
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Fusión de Membrana/fisiología , Proteínas SNARE/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Citometría de Flujo , Microscopía Fluorescente , Transfección , beta-Galactosidasa/metabolismoRESUMEN
The extent and temporal characteristics of G protein-coupled receptor (GPCR) signaling are shaped by the regulator of G protein signaling (RGS) proteins, which promote G protein deactivation. With hundreds of GPCRs and dozens of RGS proteins, compartmentalization plays a key role in establishing signaling specificity. However, the molecular details and mechanisms of this process are poorly understood. In this paper, we report that the R7 group of RGS regulators is controlled by interaction with two previously uncharacterized orphan GPCRs: GPR158 and GPR179. We show that GPR158/179 recruited RGS complexes to the plasma membrane and augmented their ability to regulate GPCR signaling. The loss of GPR179 in a mouse model of night blindness prevented targeting of RGS to the postsynaptic compartment of bipolar neurons in the retina, illuminating the role of GPR179 in night vision. We propose that the interaction of RGS proteins with orphan GPCRs promotes signaling selectivity in G protein pathways.
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Proteínas de Unión al GTP/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Animales , Comunicación Celular , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Inmunoprecipitación , Espectrometría de Masas , Ratones , Microscopía Confocal , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , TransfecciónRESUMEN
Integrins are the primary receptors of cells adhering to the extracellular matrix, and play key roles in various cellular processes including migration, proliferation and survival. The expression and distribution of integrins at the cell surface is controlled by endocytosis and recycling. The present study examines the function of syntaxin 6 (STX6), a t-SNARE located in the trans-Golgi network, in integrin trafficking. STX6 is overexpressed in many types of human cancer. We show that depletion of STX6 inhibits chemotactic cell migration and the delivery of the laminin receptor α3ß1 integrin to the cell surface, whereas STX6 overexpression stimulates chemotactic cell migration, integrin delivery, and integrin-initiated activation of focal adhesion kinase. These data indicate that STX6 plays a rate-limiting role in cell migration and integrin trafficking. In STX6-depleted cells, α3ß1 integrin is accumulated in recycling endosomes that contain the v-SNARE VAMP3. Importantly, we show that STX6 and VAMP3 form a v-/t-SNARE complex, VAMP3 is required in α3ß1 integrin delivery to the cell surface, and endocytosed α3ß1 integrin traffics to both VAMP3 and STX6 compartments. Collectively, our data suggest a new integrin trafficking pathway in which endocytosed integrins are transported from VAMP3-containing recycling endosomes to STX6-containing trans-Golgi network before being recycled to the plasma membrane.
Asunto(s)
Quimiotaxis/fisiología , Integrinas/metabolismo , Proteínas Qa-SNARE/biosíntesis , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Adhesión Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Endosomas/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Transporte de Proteínas , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Transfección , Proteína 3 de Membrana Asociada a Vesículas/genéticaRESUMEN
Integrins, a family of heterodimeric receptors for cell adhesion to the extracellular matrix (ECM), play key roles in cell migration, cancer progression and metastasis. As transmembrane proteins, integrins are transported in vesicles and delivered to the cell surface by vesicular trafficking. The final step for integrin delivery, i.e., fusion of integrin-containing vesicles with the plasma membrane, is poorly understood at the molecular level. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxins 1, 2, 3 and 4 are present at the plasma membrane to drive vesicle fusion. In this study, we examined the roles of syntaxins 1, 2, 3 and 4 in vesicular trafficking of α5ß1 and α3ß1 integrins. We showed that syntaxins 2, 3 and 4 were expressed in HeLa cervical adenocarcinoma cells and PANC-1 pancreatic adenocarcinoma cells. In migrating HeLa and PANC-1 cells, syntaxins 2, 3 and 4 co-localized with the lipid raft constituent GM1 ganglioside at the leading edge. siRNA knockdown (KD) of syntaxins 3 and 4, but not of syntaxin 2, in HeLa cells reduced cell surface expression of α5ß1 and α3ß1 integrins and accumulated the integrins in cytoplasmic vesicles, indicating that syntaxins 3 and 4 mediate vesicular trafficking of α5ß1 and α3ß1 integrins to the cell surface. In addition, KD of syntaxins 3 and 4 inhibited cell adhesion to fibronectin, suppressed chemotactic cell migration and triggered apoptosis. Collectively, these data suggest that syntaxins 3- and 4-dependent integrin trafficking is important in cancer cell migration and survival, and may be a valuable target for cancer therapy.
Asunto(s)
Movimiento Celular/fisiología , Integrina alfa3beta1/metabolismo , Integrina alfa5beta1/metabolismo , Neoplasias/patología , Proteínas Qa-SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Apoptosis/fisiología , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Fibronectinas/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Células HeLa , Humanos , Integrina alfa3beta1/antagonistas & inhibidores , Integrina alfa5beta1/antagonistas & inhibidores , Microdominios de Membrana/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Transporte de Proteínas , Seudópodos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
BACKGROUND: Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of ß-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30-50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction. CONCLUSIONS/SIGNIFICANCE: These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.
Asunto(s)
Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Animales , Células COS , Fusión Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Células Endoteliales/citología , Aparato de Golgi/metabolismo , Humanos , Operón Lac , Proteínas de la Membrana/metabolismoRESUMEN
Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of alpha5beta1 integrin. VAMP2 was present on vesicles containing endocytosed beta1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface alpha5beta1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of alpha5beta1, without altering cell surface expression of alpha2beta1 integrin or alpha3beta1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of alpha5beta1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.
Asunto(s)
Membrana Celular/metabolismo , Integrina alfa5beta1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Apoptosis/fisiología , Adhesión Celular/fisiología , Proliferación Celular , Quimiotaxis/fisiología , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Combinación de Medicamentos , Fibronectinas/metabolismo , Fibronectinas/farmacología , Células HeLa , Humanos , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Laminina/metabolismo , Transporte de Proteínas , Proteoglicanos/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , ARN Interferente Pequeño/genética , Vesículas Transportadoras/metabolismoRESUMEN
Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of beta1 integrin at the cell surface but had no effect on total cellular beta1 integrin, indicating that VAMP3 is required for trafficking of beta1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.