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1.
Nucleic Acids Res ; 50(D1): D678-D686, 2022 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-34469532

RESUMEN

Olfaction is a multi-stage process that initiates with the odorants entering the nose and terminates with the brain recognizing the odor associated with the odorant. In a very intricate way, the process incorporates various components functioning together and in synchronization. OlfactionBase is a free, open-access web server that aims to bring together knowledge about many aspects of the olfaction mechanism in one place. OlfactionBase contains detailed information of components like odors, odorants, and odorless compounds with physicochemical and ADMET properties, olfactory receptors (ORs), odorant- and pheromone binding proteins, OR-odorant interactions in Human and Mus musculus. The dynamic, user-friendly interface of the resource facilitates exploration of different entities: finding chemical compounds having desired odor, finding odorants associated with OR, associating chemical features with odor and OR, finding sequence information of ORs and related proteins. Finally, the data in OlfactionBase on odors, odorants, olfactory receptors, human and mouse OR-odorant pairs, and other associated proteins could aid in the inference and improved understanding of odor perception, which might provide new insights into the mechanism underlying olfaction. The OlfactionBase is available at https://bioserver.iiita.ac.in/olfactionbase/.


Asunto(s)
Bases de Datos Factuales , Odorantes , Neuronas Receptoras Olfatorias/química , Receptores Odorantes/genética , Animales , Humanos , Ratones , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/química , Transducción de Señal/genética , Olfato/genética
2.
J Neurochem ; 159(6): 1028-1044, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34359098

RESUMEN

Modulation of sensory perception by homeostatic feedback from physiological states is central to innate purposive behaviors. Olfaction is an important predictive modality for feeding-related behaviors and its modulation has been associated with hunger-satiety states. However, the mechanisms mapping internal states to chemosensory processing in order to modify behavior are poorly understood. In the zebrafish olfactory epithelium, a subset of olfactory sensory neurons (OSNs) and the terminal nerve projections express neuropeptide Y (NPY). Using a combination of neuronal activity and behavioral evaluation, we find that NPY signaling in the peripheral olfactory system of zebrafish is correlated with its nutritional state and is both necessary and sufficient for the olfactory perception of food-related odorants. NPY activity dynamically modulates the microvillar OSN activation thresholds and acts cooperatively with amino acid signaling resulting in a switch-like increase in OSN sensitivity in starved animals. We suggest that cooperative activation of phospholipase C by convergent signaling from NPY and amino acid receptors is central to this heightened sensitivity. This study provides ethologically relevant, physiological evidence for NPY signaling in the modulation of OSN sensitivity to food-associated amino acid cues. We demonstrate sensory gating directly at the level of OSNs and identify a novel mechanistic framework for tuning olfactory sensitivity to prevailing energy states. Cover Image for this issue: https://doi.org/10.1111/jnc.15091.


Asunto(s)
Señales (Psicología) , Ingestión de Alimentos/fisiología , Neuropéptido Y/biosíntesis , Estado Nutricional/fisiología , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Animales , Animales Modificados Genéticamente , Femenino , Humanos , Masculino , Neuropéptido Y/análisis , Mucosa Olfatoria/química , Neuronas Receptoras Olfatorias/química , Pez Cebra
3.
J Neurosci ; 41(6): 1218-1241, 2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33402421

RESUMEN

Critical periods are developmental windows during which neural circuits effectively adapt to the new sensory environment. Animal models of fragile X syndrome (FXS), a common monogenic autism spectrum disorder (ASD), exhibit profound impairments of sensory experience-driven critical periods. However, it is not known whether the causative fragile X mental retardation protein (FMRP) acts uniformly across neurons, or instead manifests neuron-specific functions. Here, we use the genetically-tractable Drosophila brain antennal lobe (AL) olfactory circuit of both sexes to investigate neuron-specific FMRP roles in the odorant experience-dependent remodeling of the olfactory sensory neuron (OSN) innervation during an early-life critical period. We find targeted OSN class-specific FMRP RNAi impairs innervation remodeling within AL synaptic glomeruli, whereas global dfmr1 null mutants display relatively normal odorant-driven refinement. We find both OSN cell autonomous and cell non-autonomous FMRP functions mediate odorant experience-dependent remodeling, with AL circuit FMRP imbalance causing defects in overall glomerulus innervation refinement. We find OSN class-specific FMRP levels bidirectionally regulate critical period remodeling, with odorant experience selectively controlling OSN synaptic terminals in AL glomeruli. We find OSN class-specific FMRP loss impairs critical period remodeling by disrupting responses to lateral modulation from other odorant-responsive OSNs mediating overall AL gain control. We find that silencing glutamatergic AL interneurons reduces OSN remodeling, while conversely, interfering with the OSN class-specific GABAA signaling enhances remodeling. These findings reveal control of OSN synaptic remodeling by FMRP with neuron-specific circuit functions, and indicate how neural circuitry can compensate for global FMRP loss to reinstate normal critical period brain circuit remodeling.SIGNIFICANCE STATEMENT Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism spectrum disorder (ASD), manifests severe neurodevelopmental delays. Likewise, FXS disease models display disrupted neurodevelopmental critical periods. In the well-mapped Drosophila olfactory circuit model, perturbing the causative fragile X mental retardation protein (FMRP) within a single olfactory sensory neuron (OSN) class impairs odorant-dependent remodeling during an early-life critical period. Importantly, this impairment requires activation of other OSNs, and the olfactory circuit can compensate when FMRP is removed from all OSNs. Understanding the neuron-specific FMRP requirements within a developing neural circuit, as well as the FMRP loss compensation mechanisms, should help us engineer FXS treatments. This work suggests FXS treatments could use homeostatic mechanisms to alleviate circuit-level deficits.


Asunto(s)
Período Crítico Psicológico , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Corteza Olfatoria/crecimiento & desarrollo , Corteza Olfatoria/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Masculino , Plasticidad Neuronal/efectos de los fármacos , Neuronas/química , Neuronas/efectos de los fármacos , Odorantes , Bulbo Olfatorio/química , Bulbo Olfatorio/metabolismo , Corteza Olfatoria/química , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/metabolismo , Optogenética/métodos
5.
Sci Rep ; 10(1): 9064, 2020 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-32493940

RESUMEN

A bioelectronic nose device based on micelle-stabilized olfactory receptors is developed for the selective discrimination of a butter flavor substance in commercial fermented alcoholic beverages. In this work, we have successfully overexpressed ODR-10, a type of olfactory receptor, from Caenorhabditis elegans using a bacterial expression system at a low cost and high productivity. The highly-purified ODR-10 was stabilized in micelle structures, and it was immobilized on a carbon nanotube field-effect transistor to build a bioelectronic nose for the detection of diacetyl, a butter flavor substance, via the specific interaction between diacetyl and ODR-10. The bioelectronic nose device can sensitively detect diacetyl down to 10 fM, and selectively discriminate it from other substances. In addition, this sensor could directly evaluate diacetyl levels in a variety of real fermented alcoholic beverages such as beer, wine, and makgeolli (fermented Korean wine), while the sensor did not respond to soju (Korean style liquor without diacetyl). In this respect, our sensor should be a powerful tool for versatile food industrial applications such as the quality control of alcoholic beverages and foods.


Asunto(s)
Bebidas Alcohólicas/análisis , Nariz Electrónica , Alimentos Fermentados/análisis , Aromatizantes/química , Neuronas Receptoras Olfatorias/química , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Animales , Técnicas Biosensibles/métodos , Mantequilla , Caenorhabditis elegans/metabolismo , Diacetil/química , Células HEK293 , Humanos , Micelas , Nanotubos de Carbono/química , Nariz/fisiología , Odorantes/análisis , Olfato/fisiología , Transistores Electrónicos
6.
J Comp Neurol ; 528(13): 2239-2253, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32080843

RESUMEN

Individual receptor neurons in the peripheral olfactory organ extend long axons into the olfactory bulb forming synapses with projection neurons in spherical neuropil regions, called glomeruli. Generally, odor map formation and odor processing in all vertebrates is based on the assumption that receptor neuron axons exclusively connect to a single glomerulus without any axonal branching. We comparatively tested this hypothesis in multiple fish and amphibian species (both sexes) by applying sparse cell electroporation to trace single olfactory receptor neuron axons. Sea lamprey (jawless fish) and zebrafish (bony fish) support the unbranched axon concept, with 94% of axons terminating in single glomeruli. Contrastingly, axonal projections of the axolotl (salamander) branch extensively before entering up to six distinct glomeruli. Receptor neuron axons labeled in frog species (Pipidae, Bufonidae, Hylidae, and Dendrobatidae) predominantly bifurcate before entering a glomerulus and 59 and 50% connect to multiple glomeruli in larval and postmetamorphotic animals, respectively. Independent of developmental stage, lifestyle, and adaptations to specific habitats, it seems to be a common feature of amphibian olfactory receptor neuron axons to frequently bifurcate and connect to multiple glomeruli. Our study challenges the unbranched axon concept as a universal vertebrate feature and it is conceivable that also later diverging vertebrates deviate from it. We propose that this unusual wiring logic evolved around the divergence of the terrestrial tetrapod lineage from its aquatic ancestors and could be the basis of an alternative way of odor processing.


Asunto(s)
Neuronas Receptoras Olfatorias/fisiología , Ambystoma mexicanum , Anfibios , Animales , Bufo marinus , Femenino , Masculino , Neuronas Receptoras Olfatorias/química , Petromyzon , Especificidad de la Especie , Xenopus
7.
Proc Natl Acad Sci U S A ; 117(6): 2957-2967, 2020 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-31974307

RESUMEN

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.


Asunto(s)
Receptores Odorantes/química , Animales , Línea Celular , Humanos , Ratones , Simulación de Dinámica Molecular , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/metabolismo , Estabilidad Proteica , Receptores Odorantes/genética , Receptores Odorantes/metabolismo
8.
PLoS One ; 15(1): e0217665, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31923248

RESUMEN

Olfactory sense remains elusive regarding the primary reception mechanism. Some studies suggest that olfaction is a spectral sense, the olfactory event is triggered by electron transfer (ET) across the odorants at the active sites of odorant receptors (ORs). Herein we present a Donor-Bridge-Acceptor model, proposing that the ET process can be viewed as an electron hopping from the donor molecule to the odorant molecule (Bridge), then hopping off to the acceptor molecule, making the electronic state of the odorant molecule change along with vibrations (vibronic transition). The odorant specific parameter, Huang-Rhys factor can be derived from ab initio calculations, which make the simulation of ET spectra achievable. In this study, we revealed that the emission spectra (after Gaussian convolution) can be acted as odor characteristic spectra. Using the emission spectrum of ET, we were able to reasonably interpret the similar bitter-almond odors among hydrogen cyanide, benzaldehyde and nitrobenzene. In terms of isotope effects, we succeeded in explaining why subjects can easily distinguish cyclopentadecanone from its fully deuterated analogue cyclopentadecanone-d28 but not distinguishing acetophenone from acetophenone-d8.


Asunto(s)
Percepción Olfatoria/genética , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/genética , Olfato/genética , Benzaldehídos/farmacología , Respiración de la Célula/genética , Transporte de Electrón/genética , Humanos , Cianuro de Hidrógeno/farmacología , Isótopos/farmacología , Nitrobencenos/farmacología , Odorantes/análisis , Percepción Olfatoria/fisiología , Neuronas Receptoras Olfatorias/química , Receptores Odorantes/metabolismo , Olfato/fisiología , Vibración
9.
PLoS One ; 14(7): e0220259, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31339957

RESUMEN

The olfactory epithelium is continuously exposed to exogenous chemicals, including odorants. During the past decade, the enzymes surrounding the olfactory receptors have been shown to make an important contribution to the process of olfaction. Mammalian xenobiotic metabolizing enzymes, such as cytochrome P450, esterases and glutathione transferases (GSTs), have been shown to participate in odorant clearance from the olfactory receptor environment, consequently contributing to the maintenance of sensitivity toward odorants. GSTs have previously been shown to be involved in numerous physiological processes, including detoxification, steroid hormone biosynthesis, and amino acid catabolism. These enzymes ensure either the capture or the glutathione conjugation of a large number of ligands. Using a multi-technique approach (proteomic, immunocytochemistry and activity assays), our results indicate that GSTs play an important role in the rat olfactory process. First, proteomic analysis demonstrated the presence of different putative odorant metabolizing enzymes, including different GSTs, in the rat nasal mucus. Second, GST expression was investigated in situ in rat olfactory tissues using immunohistochemical methods. Third, the activity of the main GST (GSTM2) odorant was studied with in vitro experiments. Recombinant GSTM2 was used to screen a set of odorants and characterize the nature of its interaction with the odorants. Our results support a significant role of GSTs in the modulation of odorant availability for receptors in the peripheral olfactory process.


Asunto(s)
Glutatión Transferasa/análisis , Moco/química , Mucosa Olfatoria/química , Secuencia de Aminoácidos , Animales , Glutatión Transferasa/metabolismo , Inmunohistoquímica , Masculino , Moco/metabolismo , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/metabolismo , Proteómica , Ratas , Ratas Wistar , Sistema Respiratorio/química , Sistema Respiratorio/metabolismo
10.
J Neurosci ; 38(43): 9240-9251, 2018 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-30201774

RESUMEN

Odorants are coded in the primary olfactory processing centers by spatially and temporally distributed patterns of glomerular activity. Whereas the spatial distribution of odorant-induced responses is known to be conserved across individuals, the universality of its temporal structure is still debated. Via fast two-photon calcium imaging, we analyzed the early phase of neuronal responses in the form of the activity onset latencies in the antennal lobe projection neurons of honeybee foragers. We show that each odorant evokes a stimulus-specific response latency pattern across the glomerular coding space. Moreover, we investigate these early response features for the first time across animals, revealing that the order of glomerular firing onsets is conserved across individuals and allows them to reliably predict odorant identity, but not concentration. These results suggest that the neuronal response latencies provide the first available code for fast odor identification.SIGNIFICANCE STATEMENT Here, we studied early temporal coding in the primary olfactory processing centers of the honeybee brain by fast imaging of glomerular responses to different odorants across glomeruli and across individuals. Regarding the elusive role of rapid response dynamics in olfactory coding, we were able to clarify the following aspects: (1) the rank of glomerular activation is conserved across individuals, (2) its stimulus prediction accuracy is equal to that of the response amplitude code, and (3) it contains complementary information. Our findings suggest a substantial role of response latencies in odor identification, anticipating the static response amplitude code.


Asunto(s)
Odorantes , Vías Olfatorias/fisiología , Neuronas Receptoras Olfatorias/fisiología , Tiempo de Reacción/fisiología , Olfato/fisiología , Animales , Abejas , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Vías Olfatorias/química , Vías Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/efectos de los fármacos , Tiempo de Reacción/efectos de los fármacos , Olfato/efectos de los fármacos
11.
Elife ; 72018 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-30080137

RESUMEN

Centrioles are cylindrical assemblies comprised of 9 singlet, doublet, or triplet microtubules, essential for the formation of motile and sensory cilia. While the structure of the cilium is being defined at increasing resolution, centriolar structure remains poorly understood. Here, we used electron cryo-tomography to determine the structure of mammalian (triplet) and Drosophila (doublet) centrioles. Mammalian centrioles have two distinct domains: a 200 nm proximal core region connected by A-C linkers, and a distal domain where the C-tubule is incomplete and a pair of novel linkages stabilize the assembly producing a geometry more closely resembling the ciliary axoneme. Drosophila centrioles resemble the mammalian core, but with their doublet microtubules linked through the A tubules. The commonality of core-region length, and the abrupt transition in mammalian centrioles, suggests a conserved length-setting mechanism. The unexpected linker diversity suggests how unique centriolar architectures arise in different tissues and organisms.


Asunto(s)
Centriolos/ultraestructura , Cilios/ultraestructura , Microscopía por Crioelectrón , Neuronas Receptoras Olfatorias/ultraestructura , Animales , Células CHO , Centriolos/química , Cilios/química , Cricetulus , Drosophila melanogaster , Tomografía con Microscopio Electrónico , Microtúbulos/química , Microtúbulos/ultraestructura , Neuronas Receptoras Olfatorias/química
13.
Microsc Res Tech ; 78(7): 613-9, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25950169

RESUMEN

Little is known about the development of the olfactory organs of camel. In this study, prenatal development and neuronal differentiation of the vomeronasal organ (VNO) and the olfactory epithelium (OE) of the one-humped camel were studied by immunohistochemistry and lectin histochemistry. A neuronal marker, protein gene product (PGP) 9.5, but not a marker of fully differentiated olfactory receptor cells, olfactory marker protein, intensely labeled the olfactory receptor cells of the VNO and OE at 395 mm, 510 mm, and 530 mm fetal ages, indicating that the olfactory receptor cells are differentiated, but not fully matured both in the VNO and the OE. In 187 mm and 190 mm fetuses, PGP 9.5 yielded faint immunoreactive signals in the VNO, but not in the OE, although the presence of olfactory receptor cells were demonstrated in both tissues by intense WGA and LEL stainings. We conclude that the camel VNO and OE bear differentiated, but still immature receptor cells; in addition, the onset of neuronal differentiation seems to be somewhat earlier in the VNO than in the OE till half of the prenatal life.


Asunto(s)
Camelus/embriología , Lectinas/metabolismo , Organogénesis , Órgano Vomeronasal/química , Órgano Vomeronasal/embriología , Animales , Camelus/metabolismo , Diferenciación Celular , Femenino , Inmunohistoquímica , Lectinas/análisis , Masculino , Mucosa Olfatoria/química , Mucosa Olfatoria/embriología , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/metabolismo , Órgano Vomeronasal/metabolismo
14.
J Vis Exp ; (87)2014 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-24834898

RESUMEN

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.


Asunto(s)
Antenas de Artrópodos/química , Antenas de Artrópodos/citología , Técnicas Inmunológicas/métodos , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/citología , Animales , Drosophila
15.
J Vis Exp ; (94)2014 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-25590569

RESUMEN

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.


Asunto(s)
Cilios/química , Bulbo Olfatorio/química , Neuronas Receptoras Olfatorias/química , Células Receptoras Sensoriales/química , Coloración y Etiquetado/métodos , Animales , Cilios/metabolismo , Inmunoglobulinas/química , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo
16.
Angew Chem Int Ed Engl ; 52(42): 11021-4, 2013 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-24038729

RESUMEN

Receptor-ligand interaction: Olfactory receptors (ORs) are G-protein-coupled receptors (GPCRs), which detect signaling molecules such as hormones and odorants. The structure of opsin, the GPCR employed in vision, with a detergent molecule bound deep in its orthosteric ligand-binding pocket provides a template for OR homology modeling, thus enabling investigation of the structural basis of the mechanism of odorant-receptor recognition.


Asunto(s)
Neuronas Receptoras Olfatorias/química , Opsinas/química , Receptores Acoplados a Proteínas G/química , Humanos , Modelos Moleculares , Neuronas Receptoras Olfatorias/metabolismo , Opsinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
17.
Biochimie ; 95(10): 1909-16, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23816872

RESUMEN

Although the identification of the multigene family encoding mammalian olfactory receptors were identified more than 20 years ago, we are far from understanding olfactory perception because of the difficulties in functional expression of these receptors in heterologous cell systems. Cell-free (CF) or in vitro expression systems offer an elegant alternative route to cell based protein expression, as the functional expression of membrane proteins can be directly achieved from the genetic template without the need of cell cultivation and protein isolation. Here we investigated in detail the cell-free expression and membrane insertion of the olfactory receptor OR5 in dependence of different experimental conditions like probing different origins of the cell-free expression system (from bacteria, via plants and insects toward mammalian system) and lipid composition of the respective extracts. We provided substantial biochemical indications by radioactive labeling based on [(35)S]-methionine, followed by proteolytic digestion, and we found that the insertion of the olfactory receptor OR5 into liposomes resulted in an unidirectional orientation with the binding side exposed into the aqueous space, resembling the native orientation in the cilia of the olfactory neurons. We report the different results in synthesis capacity for the different in vitro systems employed as we like to demonstrate the first in vitro kit toward and ex situ and ex vivo odorant receptor array.


Asunto(s)
Expresión Génica , Neuronas Receptoras Olfatorias/química , Biosíntesis de Proteínas/genética , Receptores Odorantes/genética , Liposomas Unilamelares/metabolismo , Animales , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Metionina/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Estructura Terciaria de Proteína , Proteolisis , Conejos , Ratas , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Reticulocitos/química , Reticulocitos/metabolismo , Radioisótopos de Azufre , Liposomas Unilamelares/química
18.
Insect Biochem Mol Biol ; 43(2): 138-45, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23196131

RESUMEN

Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit. The integral membrane proteins SNMP1 and 2 are also associated with olfactory function, with SNMP1 required for cis-vaccinyl acetate reception in Drosophila melanogaster. In order to investigate protein-protein interactions among these membrane proteins we measured intermolecular Förster/Fluorescence Resonance Energy Transfer (FRET) in live insect cells by acceptor photobleaching. Fusion proteins containing Cyan Fluorescent Protein or Yellow Fluorescent Protein were produced using baculovirus-mediated expression in High Five™ cells. The majority of the recombinant products were of the expected size for the fusion proteins and located within intracellular membranes. We were able to show FRET efficiencies providing evidence for homomeric and heteromeric interactions of the ligand-binding OR, Or22a, and Orco (Or22a-Or22a, Or22a-Orco, Orco-Orco). There was no evidence for an interaction between SNMP1 and Orco or between SNMP2 and Orco or Or22a. However, fusion proteins of SNMP1 and Or22a did show an interaction by FRET, suggesting SNMP1 may interact with at least some insect olfactory receptor complexes. In summary, this study supports previously observed homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a, and identifies a novel interaction between Or22a and SNMP1.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores Odorantes/metabolismo , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/metabolismo , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Odorantes/química , Receptores Odorantes/genética
19.
Biosens Bioelectron ; 42: 570-80, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23261691

RESUMEN

The biological olfactory system can recognize and discriminate thousands of volatile organic compounds (VOCs) with extremely high sensitivity and specificity. The most fundamental elements are olfactory receptors (ORs) in the cilia of olfactory sensory neurons (OSNs), which contribute greatly to the high-performance olfactory system. The excellent properties of ORs are generally recognized in the development of biomimetic OR-based biosensors. Over the past two decades, much work has been done in developing OR-based biosensors due to their promising potential in many applications. In this article, we will outline the latest advances of OR-based biosensors. Two current crucial issues in this field will be discussed, namely, the production methods and immobilization techniques of ORs. We will also elaborate on various OR-based biosensors and their latest developments. Finally, current research trends and future challenges in this field will be discussed.


Asunto(s)
Técnicas Biosensibles/métodos , Neuronas Receptoras Olfatorias , Receptores Odorantes , Animales , Cilios/fisiología , Humanos , Neuronas Aferentes/química , Neuronas Aferentes/fisiología , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/fisiología , Receptores Odorantes/química , Receptores Odorantes/genética , Receptores Odorantes/fisiología , Sensibilidad y Especificidad , Olfato
20.
J Neurosci ; 32(40): 13819-40, 2012 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-23035093

RESUMEN

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.


Asunto(s)
Señalización del Calcio , Colorantes Fluorescentes/química , Fluorometría/métodos , Proteínas Fluorescentes Verdes/química , Neuroimagen/métodos , Neuronas/química , Péptidos/química , Transmisión Sináptica , Animales , Astrocitos/química , Astrocitos/ultraestructura , Caenorhabditis elegans , Cristalografía por Rayos X , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Colorantes Fluorescentes/análisis , Genes Sintéticos , Vectores Genéticos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Células HEK293/química , Células HEK293/ultraestructura , Hipocampo/química , Hipocampo/citología , Humanos , Larva , Rayos Láser , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Neuromuscular/química , Unión Neuromuscular/ultraestructura , Neuronas/fisiología , Neuronas/ultraestructura , Neurópilo/química , Neurópilo/fisiología , Neurópilo/ultraestructura , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/fisiología , Neuronas Receptoras Olfatorias/ultraestructura , Péptidos/análisis , Péptidos/genética , Estimulación Luminosa , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Células Bipolares de la Retina/química , Células Bipolares de la Retina/fisiología , Células Bipolares de la Retina/ultraestructura , Pez Cebra/crecimiento & desarrollo
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