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Robert A. Arkowitz - One of the best experts on this subject based on the ideXlab platform.
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In Situ Assays of Chemotropism During Yeast Mating
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: David E. Stone, Robert A. ArkowitzAbstract:Virtually all eukaryotic cells can grow in a polarized fashion in response to external signals. Cells can respond to gradients of chemoattractants or chemorepellents by directional growth, a process referred to as Chemotropism. The budding yeast Saccharomyces cerevisiae undergoes chemotropic growth during mating, in which two haploid cells of opposite mating type grow towards one another. Mating pheromone gradients are essential for efficient mating in yeast and different yeast mutants are defective in Chemotropism. Two methods of assessing the ability of yeast strains to respond to pheromone gradients are presented here.
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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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Phosphorylation of Gβ is crucial for efficient Chemotropism in yeast.
Journal of Cell Science, 2013Co-Authors: Reagan Deflorio, Nicholas Waszczak, Metodi V. Metodiev, Robert A. Arkowitz, Marie-elena Brett, Elisabetta Apollinari, Oleksii Dubrovskyi, David Eddington, David E. StoneAbstract:Mating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Gα dissociates from Gβγ and Gβ is subsequently phosphorylated. Free Gβγ signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gβγ-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gβ that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gβ plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie growth site selection and maintenance.
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A Cdc24p-Far1p-G� � Protein Complex Required for Yeast Orientation during Mating
2013Co-Authors: Aljoscha Nern, Robert A. ArkowitzAbstract:Abstract. Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During mating, haploid Saccharomyces cerevisiae cells orient their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the bud site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here we show that a twohybrid interaction between Cdc24p and G � requires Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and G�. Binding experiments demonstrate that Cdc24p, Far1p, and G � form a complex in which pairwise interactions can occur in the absence of the third protein. Cdc24p localizes to sites of polarized growth suggesting that this complex is localized. In the absence of CDC24-FAR1-mediated Chemotropism, a bud site selection protein, Bud1p/Rsr1p, is essential for morphological changes in response to pheromone. These results suggest that formation of a Cdc24p-Far1p-G� � complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal. Key words: Chemotropism • landmark • oriented growth • Ste4p Ste18p • yeast mating EUKARYOTIC cells are able to polarize their growth in response to both external and internal signals. Polarization to external signals plays a crucial role in development and tissue formation. During yeast mating, cells of opposite mating type secrete peptide pheromones and respond to pheromone from their mating partner (for review see Sprague and Thorner, 1992; Chenevert, 1994; Leberer et al., 1997a). Mating pheromone binds to specific G-protein–coupled receptors on cells of opposite mating type (Bender and Sprague, 1989; Blumer et al., 1988). Receptor activation results in cell cycle arrest, transcriptional activation, morphological changes, and polarized growth towards a partner cell (Sprague and Thorner, 1992; Chenevert, 1994; Leberer et al., 1997a). Cells respond to a gradient of mating pheromone by oriented growth along this gradient (Segall, 1993). Such chemotropic growth is essential for efficient mating (Dore
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Brief Report Nucleocytoplasmic Shuttling of the Cdc42p Exchange Factor Cdc24p
2013Co-Authors: Aljoscha Nern, Robert A. ArkowitzAbstract:Abstract. Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle–dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p–Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p– Far1p complex which is required for Chemotropism. Key words: nucleocytoplasmic shuttling • GDP/GTP exchange factor • Far1p • Cdc24p • polarized growt
Leland H. Hartwell - One of the best experts on this subject based on the ideXlab platform.
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Mating in Saccharomyces cerevisiae: The Role of the Pheromone Signal Transduction Pathway in the Chemotropic Response to Pheromone
Genetics, 1997Co-Authors: Kathrin Schrick, Barbara Garvik, Leland H. HartwellAbstract:The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is Chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in Chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for Chemotropism. In contrast, cells mutant for the receptor (ste2) or the beta or gamma subunit (ste4 and ste18) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for Chemotropism.
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Saccharomyces cerevisiae cells execute a default pathway to select a mate in the absence of pheromone gradients
The Journal of cell biology, 1995Co-Authors: Russell Dorer, Peter M. Pryciak, Leland H. HartwellAbstract:During conjugation, haploid S. cerevisiae cells find one another by polarizing their growth toward each other along gradients of pheromone (Chemotropism). We demonstrate that yeast cells exhibit a second mating behavior: when their receptors are saturated with pheromone, wild-type a cells execute a default pathway and select a mate at random. These matings are less efficient than chemotropic matings, are induced by the same dose of pheromone that induces shmoo formation, and appear to use a site near the incipient bud site for polarization. We show that the SPA2 gene is specifically required for the default pathway: spa2 delta mutants cannot mate if pheromone concentrations are high and gradients are absent, but can mate if gradients are present. ste2 delta, sst2 delta, and far1 delta mutants are Chemotropism-defective and therefore must choose a mate by using a default pathway; consistent with this deduction, these strains require SPA2 to mate. In addition, our results suggest that far1 mutants are Chemotropism-defective because their mating polarity is fixed at the incipient bud site, suggesting that the FAR1 gene is required for inhibiting the use of the incipient bud site during chemotropic mating. These observations reveal a molecular relationship between the mating and budding polarity pathways.
Elizabeth M. Lord - One of the best experts on this subject based on the ideXlab platform.
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Pollen tube guidance: the role of adhesion and chemotropic molecules.
Current topics in developmental biology, 2004Co-Authors: Sunran Kim, Juan Dong, Elizabeth M. LordAbstract:Publisher Summary In many reproductive systems, adhesion and guidance are essential components of fertilization. In animals, sperm cells are motile and when guided to the egg cell, the sperm penetrates through the extracellular matrix (ECM) and adheres to the egg plasma membrane. Sperm cells in marine animals and algae are released into the open sea and must be guided to the egg. Marine invertebrates provide excellent material for the study of fertilization and information about the basic biology of the interaction between cell surfaces, as well as discoveries of the few molecules known to be involved in sperm–egg interaction. Motile sperm cells are also present in the lower divisions of land plants and in some seed plants. In ferns, sperm cells are released from the male gametophyte (gamete producing haploid phase of the life cycle) and swim, usually through rainwater, to the nearby female gametophyte, which bears archegonia with eggs enclosed. In both animals and plants, guidance cues from the female tissues are critical to accomplish fertilization. One of the most extensively studied guidance mechanisms is chemotaxis, defined as the movement of a cell up a molecular gradient; Chemotropism refers to the growth of a cell up a molecular gradient. Adhesion molecules have been implicated in guidance in both reproduction and in embryonic cell movements in animal development, including neuron guidance. In plants, adhesion molecules are involved in reproduction, both in guidance and in the fertilization event, of gamete fusion. It is not unusual to find the same molecules involved in both adhesion and Chemotropism in animals. However, in plants only a few such molecules have been reported. Recently, molecules involved in both pollen tube adhesion and guidance have been isolated from the flowering plant stigma and style transmitting tract tissues. This chapter discusses adhesion and Chemotropism and looks at a chemotropic peptide in the lily stigma.
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chemocyanin a small basic protein from the lily stigma induces pollen tube Chemotropism
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Jeanclaude Mollet, Juan Dong, Kangling Zhang, Sang Youl Park, Elizabeth M. LordAbstract:In plant reproduction, pollination is an essential process that delivers the sperm through specialized extracellular matrices (ECM) of the pistil to the ovule. Although specific mechanisms of guidance for pollen tubes through the pistil are not known, the female tissues play a critical role in this event. Many studies have documented the existence of diffusible chemotropic factors in the lily stigma that can induce pollen tube Chemotropism in vitro, but no molecules have been isolated to date. In this study, we identified a chemotropic compound from the stigma by use of biochemical methods. We purified a lily stigma protein that is active in an in vitro Chemotropism assay by using cation exchange, gel filtration, and HPLC. Tryptic digestion of the protein yielded peptides that identified the protein as a plantacyanin (basic blue protein), and this was confirmed by cloning the cDNA from the lily stigma. Plantacyanins are small cell wall proteins of unknown function. The measured molecular mass by electrospray ionization ion source MS is 9,898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9,900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma/stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style.
Metodi V. Metodiev - One of the best experts on this subject based on the ideXlab platform.
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Quantitative proteomics reveals a Gα/MAPK signaling hub that controls pheromone-induced cellular polarization in yeast
Journal of proteomics, 2019Co-Authors: Nicholas Waszczak, David E. Stone, Reagan Deflorio, Amber Ismael, Naiyuan Cheng, Metodi V. MetodievAbstract:The mating-specific yeast Gα controls pheromone signaling by sequestering Gβγ and by regulating the Fus3 MAP kinase. Disrupting Gα-Fus3 interaction leads to severe defects in Chemotropism. Because Gα concentrates at the chemotropic growth site where Fus3 is required for the phosphorylation of two known targets, we screened for additional proteins whose phosphorylation depends on pheromone stimulation and Gα-Fus3 interaction. Using a mutant form of Gα severely defective in Fus3-binding, GαDSD, and quantitative mass spectrometry, fourteen proteins were identified as potential targets of Gα-recruited Fus3, ten of which were previously implicated in cell polarity and morphogenesis. To explore the biological relevance of these findings, we focused on the Spa2 polarisome protein, which was hypophosphorylated on multiple serine residues in pheromone-treated GαDSD cells. Six sites were mutagenized to create the Spa26XSA mutant protein. Spa26XSA exhibited increased affinity for Fus3, consistent with a kinase-substrate interaction, and Spa26XSA cells exhibited dramatic defects in gradient sensing and zygote formation. These results suggest that Gα promotes the phosphorylation of Spa2 by Fus3 at the cortex of pheromone-stimulated cells, and that this mechanism plays a role in Chemotropism. How the Gα-Fus3 signaling hub affects the other putative targets identified here has yet to be determined. SIGNIFICANCE: Previously, interaction between the G alpha protein, Gpa1, and the MAPK of the pheromone response pathway, Fus3, was shown to be important for efficient sensing of the pheromone gradient and for the maintenance of cell polarity during mating. Here we show that the underlying molecular mechanisms involve the phosphorylation of specific cortical targets of Gpa1/Fus3. These have been identified by quantitative phosphoproteomics using a mutant of Gpa1, which is defective in interacting with Fus3. One of these targets is the polarisome protein Spa2. Alanine substitution of the Spa2 phosphorylation sites targeted by Gpa1/Fus3 lead to a dramatic defect in pheromone gradient sensing and zygote formation. These results reveal how the G alpha protein and the MAPK control cell polarity in a prototypical model system. Our results have wider significance as similar mechanisms exist in higher eukaryotes and are involved in important biological such as neuron development, immunity, and cancer cell metastasis.
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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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Phosphorylation of Gβ is crucial for efficient Chemotropism in yeast.
Journal of Cell Science, 2013Co-Authors: Reagan Deflorio, Nicholas Waszczak, Metodi V. Metodiev, Robert A. Arkowitz, Marie-elena Brett, Elisabetta Apollinari, Oleksii Dubrovskyi, David Eddington, David E. StoneAbstract:Mating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Gα dissociates from Gβγ and Gβ is subsequently phosphorylated. Free Gβγ signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gβγ-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gβ that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gβ plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie growth site selection and maintenance.
W. Mary Lush - One of the best experts on this subject based on the ideXlab platform.
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Whither Chemotropism and pollen tube guidance
Trends in Plant Science, 1999Co-Authors: W. Mary LushAbstract:Abstract Pollen tubes follow a well-defined path to deliver male gametes to female gametes, but the mechanisms they use to locate this path are poorly understood. The major hypothesis is (and long has been) that pollen tubes are guided by chemical gradients and/or physical structures. Recently, parallels have been drawn between chemical mechanisms of guidance in pollen tubes and other cells, such as axons. These comparisons highlight a problem with the current models for pollen tube guidance, namely the distance over which chemical guidance is proposed to occur. Based on this new perspective, some models are either invalid or pollen tubes are uniquely responsive to chemical guidance cues.