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Jeremy Thorner - One of the best experts on this subject based on the ideXlab platform.
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Tracking yeast Pheromone Receptor Ste2 endocytosis using fluorogen-activating protein tagging
Molecular Biology of the Cell, 2018Co-Authors: Anita Emmerstorfer-augustin, Curlier Michel Marc Augustin, Shadi Shams, Jeremy ThornerAbstract:Author(s): Emmerstorfer-Augustin, A; Augustin, CM; Shams, S; Thorner, J | Abstract: To observe internalization of the yeast Pheromone Receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this Receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.
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tracking yeast Pheromone Receptor ste2 endocytosis using fluorogen activating protein tagging
Molecular Biology of the Cell, 2018Co-Authors: Anita Emmerstorferaugustin, Curlier Michel Marc Augustin, Shadi Shams, Jeremy ThornerAbstract:To observe internalization of the yeast Pheromone Receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this Receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.
David E Stone - One of the best experts on this subject based on the ideXlab platform.
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Polarization of the yeast Pheromone Receptor requires its internalization but not actin-dependent secretion.
Molecular Biology of the Cell, 2010Co-Authors: Dmitry V Suchkov, Amber Ismael, Robert A. Arkowitz, Reagan Deflorio, Edward Draper, Madhushalini Sukumar, David E StoneAbstract:In the best understood models of eukaryotic directional sensing, chemotactic cells maintain a uniform distribution of surface Receptors even when responding to chemical gradients. The yeast Pheromone Receptor is also uniformly distributed on the plasma membrane of vegetative cells, but Pheromone induces its polarization into "crescents" that cap the future mating projection. Here, we find that in Pheromone-treated cells, Receptor crescents are visible before detectable polarization of actin cables and that the Receptor can polarize in the absence of actin-dependent directed secretion. Receptor internalization, in contrast, seems to be essential for the generation of Receptor polarity, and mutations that deregulate this process confer dramatic defects in directional sensing. We also show that Pheromone induces the internalization and subsequent polarization of the mating-specific Galpha and Gbeta proteins and that the changes in G protein localization depend on Receptor internalization and Receptor-Galpha coupling. Our data suggest that the polarization of the Receptor and its G protein precedes actin polarization and is important for gradient sensing. We propose that the establishment of Receptor/G protein polarity depends on a novel mechanism involving differential internalization and that this serves to amplify the shallow gradient of activated Receptor across the cell.
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polarization of the yeast Pheromone Receptor requires its internalization but not actin dependent secretion
Molecular Biology of the Cell, 2010Co-Authors: Dmitry Suchkov, Amber Ismael, Robert A. Arkowitz, Reagan Deflorio, Edward Draper, Madhushalini Sukumar, David E StoneAbstract:In the best understood models of eukaryotic directional sensing, chemotactic cells maintain a uniform distribution of surface Receptors even when responding to chemical gradients. The yeast Pheromone Receptor is also uniformly distributed on the plasma membrane of vegetative cells, but Pheromone induces its polarization into “crescents” that cap the future mating projection. Here, we find that in Pheromone-treated cells, Receptor crescents are visible before detectable polarization of actin cables and that the Receptor can polarize in the absence of actin-dependent directed secretion. Receptor internalization, in contrast, seems to be essential for the generation of Receptor polarity, and mutations that deregulate this process confer dramatic defects in directional sensing. We also show that Pheromone induces the internalization and subsequent polarization of the mating-specific Gα and Gβ proteins and that the changes in G protein localization depend on Receptor internalization and Receptor–Gα coupling. Our data suggest that the polarization of the Receptor and its G protein precedes actin polarization and is important for gradient sensing. We propose that the establishment of Receptor/G protein polarity depends on a novel mechanism involving differential internalization and that this serves to amplify the shallow gradient of activated Receptor across the cell.
Robert A. Arkowitz - One of the best experts on this subject based on the ideXlab platform.
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Gβ promotes Pheromone Receptor polarization and yeast chemotropism by inhibiting Receptor phosphorylation
Science signaling, 2016Co-Authors: Amber Ismael, Nicholas Waszczak, Metodi V. Metodiev, Wei Tian, Xin Wang, Youfang Cao, Dmitry Suchkov, Eli E. Bar, Jie Liang, Robert A. ArkowitzAbstract:Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface Receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in Receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the Pheromone Receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although Receptor polarization occurs before and independently of actin cable–dependent delivery of vesicles to the plasma membrane (directed secretion), it requires Receptor internalization. Phosphorylation of Pheromone Receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the Pheromone-responsive Gβγ dimer promotes the polarization of the Pheromone Receptor by interacting with Yck1/2 and locally inhibiting Receptor phosphorylation. We also found that Receptor phosphorylation is essential for chemotropism, independently of its role in inducing Receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the Pheromone Receptor and accounts for its polarization before the initiation of directed secretion.
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Polarization of the yeast Pheromone Receptor requires its internalization but not actin-dependent secretion.
Molecular Biology of the Cell, 2010Co-Authors: Dmitry V Suchkov, Amber Ismael, Robert A. Arkowitz, Reagan Deflorio, Edward Draper, Madhushalini Sukumar, David E StoneAbstract:In the best understood models of eukaryotic directional sensing, chemotactic cells maintain a uniform distribution of surface Receptors even when responding to chemical gradients. The yeast Pheromone Receptor is also uniformly distributed on the plasma membrane of vegetative cells, but Pheromone induces its polarization into "crescents" that cap the future mating projection. Here, we find that in Pheromone-treated cells, Receptor crescents are visible before detectable polarization of actin cables and that the Receptor can polarize in the absence of actin-dependent directed secretion. Receptor internalization, in contrast, seems to be essential for the generation of Receptor polarity, and mutations that deregulate this process confer dramatic defects in directional sensing. We also show that Pheromone induces the internalization and subsequent polarization of the mating-specific Galpha and Gbeta proteins and that the changes in G protein localization depend on Receptor internalization and Receptor-Galpha coupling. Our data suggest that the polarization of the Receptor and its G protein precedes actin polarization and is important for gradient sensing. We propose that the establishment of Receptor/G protein polarity depends on a novel mechanism involving differential internalization and that this serves to amplify the shallow gradient of activated Receptor across the cell.
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polarization of the yeast Pheromone Receptor requires its internalization but not actin dependent secretion
Molecular Biology of the Cell, 2010Co-Authors: Dmitry Suchkov, Amber Ismael, Robert A. Arkowitz, Reagan Deflorio, Edward Draper, Madhushalini Sukumar, David E StoneAbstract:In the best understood models of eukaryotic directional sensing, chemotactic cells maintain a uniform distribution of surface Receptors even when responding to chemical gradients. The yeast Pheromone Receptor is also uniformly distributed on the plasma membrane of vegetative cells, but Pheromone induces its polarization into “crescents” that cap the future mating projection. Here, we find that in Pheromone-treated cells, Receptor crescents are visible before detectable polarization of actin cables and that the Receptor can polarize in the absence of actin-dependent directed secretion. Receptor internalization, in contrast, seems to be essential for the generation of Receptor polarity, and mutations that deregulate this process confer dramatic defects in directional sensing. We also show that Pheromone induces the internalization and subsequent polarization of the mating-specific Gα and Gβ proteins and that the changes in G protein localization depend on Receptor internalization and Receptor–Gα coupling. Our data suggest that the polarization of the Receptor and its G protein precedes actin polarization and is important for gradient sensing. We propose that the establishment of Receptor/G protein polarity depends on a novel mechanism involving differential internalization and that this serves to amplify the shallow gradient of activated Receptor across the cell.
Ivan Rodriguez - One of the best experts on this subject based on the ideXlab platform.
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Odorant and Pheromone Receptor gene regulation in vertebrates
Current opinion in genetics & development, 2007Co-Authors: Ivan RodriguezAbstract:The largest mammalian gene family codes for odorant Receptors and is exclusively devoted to the perception of the outside world. Its expression is very peculiar, since olfactory sensory neurons are only allowed to express a single of its numerous members, from a single parental allele. How this is achieved is unknown, but recent work points to multiple regulatory mechanisms, possibly shared by Pheromone Receptor genes, acting at (a) a general level, via the expression of the chemoReceptor itself and (b) a more restricted level, defined by activator elements.
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Remarkable diversity of mammalian Pheromone Receptor repertoires
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Ivan RodriguezAbstract:At the center of animal species survival is the ability of individuals to identify members of their own species and to discriminate between the genders of these members to procreate. This basic biological task is, in mammals, mostly mediated by the exchange of pheromonal information and performed by the olfactory system. More precisely, an elongated tubular structure, the vomeronasal organ, located in the nasal cavity and filled with sensory neurons, is mainly responsible for the detection of intraspecies chemosensory signals (1). Receptors responsible for the recognition of Pheromones (2, 3) and expressed by vomeronasal sensory neurons are G protein-coupled Receptors, termed V1r Receptors (4). The genes encoding these latter are particularly numerous in rodents (>100) and form a very diverse superfamily (5–8). In a recent issue of PNAS, Grus et al . (9) reported the identification of the V1r Pheromone Receptor repertoires pertaining to multiple orders of marsupial and placental mammals, and they observed striking variations in terms of repertoire size and content between these orders. Such extent of variability in the mammalian class for a given gene family is unusual, to say the least, and forces us to reflect on the nature of the evolutionary forces that led to such unequally distributed chemosensory tools among mammals. Mammals constitute a large group, which includes monotremes, marsupials, and eutherians, these latter comprising species apparently as unrelated as bats and whales. Our current view of the molecular chemosensory tools (i.e., the chemosensory Receptor gene repertoires) available to the different mammalian species is extremely …
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olfactory expression of a single and highly variable v1r Pheromone Receptor like gene in fish species
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Patrick Pfister, Ivan RodriguezAbstract:Sensory neurons expressing members of the seven-transmembrane V1r Receptor superfamily allow mice to perceive Pheromones. These Receptors, which exhibit no sequence homology to any known protein except a weak similarity to taste Receptors, have only been found in mammals. In the mouse, the V1r repertoire contains >150 members, which are expressed by neurons of the vomeronasal organ, a structure present exclusively in some tetrapod species. Here, we report the existence of a single V1r gene in multiple species of a non-terrestrial, vomeronasal organ-lacking taxon, the teleosts. In zebrafish, this V1r gene is expressed in chemosensory neurons of the olfactory rosette with a punctate distribution, strongly suggesting a role in chemodetection. This unique Receptor gene exhibits a remarkably high degree of sequence variability between fish species. It likely corresponds to the original V1r present in the common ancestor of vertebrates, which led to the large and very diverse expansion of vertebrate Pheromone Receptor repertoires, and suggests the presence of V1rs in multiple nonmammalian phyla.
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Pheromone Receptors in mammals
Hormones and behavior, 2004Co-Authors: Ivan RodriguezAbstract:In most mammals, Pheromone perception mediates intraspecies interactions related to reproduction, such as mate recognition, intermale aggressive behaviors, or exchanges between females and their offspring. Recent molecular findings, particularly the identification of two large Pheromone Receptor gene superfamilies, provide today invaluable tools to better understand the way mammals make sense of pheromonal information.
Takeshi Sakurai - One of the best experts on this subject based on the ideXlab platform.
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A Single Sex Pheromone Receptor Determines Chemical Response Specificity of Sexual Behavior in the Silkmoth
2016Co-Authors: Bombyx Mori, Takeshi Sakurai, Hidefumi Mitsuno, Keiro Uchino, Stephan Shuichi Haupt, Fumio YokohariAbstract:In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex Pheromones. In most moth species, male moths rely heavily on species-specific sex Pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible Pheromone system, in which a single Pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex Pheromone Receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex Pheromone Receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex Pheromone Receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex Pheromone Receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol Receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical Pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol Receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol Receptor neurons alone is sufficien
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Targeted disruption of a single sex Pheromone Receptor gene completely abolishes in vivo Pheromone response in the silkmoth
Scientific reports, 2015Co-Authors: Takeshi Sakurai, Hidefumi Mitsuno, Hideki Sezutsu, Akihisa Mikami, Keiro Uchino, Masashi Tabuchi, Feng Zhang, Ryohei KanzakiAbstract:Male moths use species-specific sex Pheromones to identify and orientate toward conspecific females. Odorant Receptors (ORs) for sex Pheromone substances have been identified as sex Pheromone Receptors in various moth species. However, direct in vivo evidence linking the functional role of these ORs with behavioural responses is lacking. In the silkmoth, Bombyx mori, female moths emit two sex Pheromone components, bombykol and bombykal, but only bombykol elicits sexual behaviour in male moths. A sex Pheromone Receptor BmOR1 is specifically tuned to bombykol and is expressed in specialized olfactory Receptor neurons (ORNs) in the Pheromone sensitive long sensilla trichodea of male silkmoth antennae. Here, we show that disruption of the BmOR1 gene, mediated by transcription activator-like effector nucleases (TALENs), completely removes ORN sensitivity to bombykol and corresponding Pheromone-source searching behaviour in male moths. Furthermore, transgenic rescue of BmOR1 restored normal behavioural responses to bombykol. Our results demonstrate that BmOR1 is required for the physiological and behavioural response to bombykol, demonstrating that it is the Receptor that mediates sex Pheromone responses in male silkmoths. This study provides the first direct evidence that a member of the sex Pheromone Receptor family in moth species mediates conspecific sex Pheromone information for sexual behaviour.
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a single sex Pheromone Receptor determines chemical response specificity of sexual behavior in the silkmoth bombyx mori
PLOS Genetics, 2011Co-Authors: Takeshi Sakurai, Hidefumi Mitsuno, Hideki Sezutsu, Takaaki Nishioka, Keiro Uchino, Stephan Shuichi Haupt, Fumio Yokohari, Isao Kobayashi, Toshiki Tamura, Ryohei KanzakiAbstract:In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex Pheromones. In most moth species, male moths rely heavily on species-specific sex Pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible Pheromone system, in which a single Pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex Pheromone Receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex Pheromone Receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex Pheromone Receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex Pheromone Receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol Receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical Pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol Receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol Receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex Pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.
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identification and functional characterization of a sex Pheromone Receptor in the silkmoth bombyx mori
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Takeshi Sakurai, Hidefumi Mitsuno, Takao Nakagawa, Hajime Mori, Yasuhisa Endo, Shintarou Tanoue, Yuji Yasukochi, Kazushige Touhara, Takaaki NishiokaAbstract:Sex Pheromones released by female moths are detected with high specificity and sensitivity in the olfactory sensilla of antennae of conspecific males. Bombykol in the silkmoth Bombyx mori was the first sex Pheromone to be identified. Here we identify a male-specific G protein-coupled olfactory Receptor gene, B. mori olfactory Receptor 1 (BmOR-1), that appears to encode a bombykol Receptor. The BmOR-1 gene is located on the Z sex chromosome, has an eight-exon/seven-intron structure, and exhibits male-specific expression in the Pheromone Receptor neurons of male moth antenna during late pupal and adult stages. Bombykol stimulation of Xenopus laevis oocytes expressing BmOR-1 and BmGαq elicited robust dose-dependent inward currents on two-electrode voltage clamp recordings, demonstrating that the binding of bombykol to BmOR-1 leads to the activation of a BmGαq-mediated signaling cascade. Antennae of female moths infected with BmOR-1-recombinant baculovirus showed electrophysiological responses to bombykol but not to bombykal. These results provide evidence that BmOR-1 is a G protein-coupled sex Pheromone Receptor that recognizes bombykol.