Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogen-activated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.Comment: This work was supported by National Institutes of Health (NIH) Predoctoral Traineeship GM07232 and a University of California at Berkeley MacArthur and Lakhan-Pal Graduate Fellowship to K.L.L., Erwin Schroedinger Fellowship J3787-B21 from the Austrian Science Fund to AE-A, Marie Sklodowska-Curie Action H2020-MSCA-IF-2016 InsiliCardio, GA 75083 to CMA, and NIH R01 research grant GM21841 to J

Classical and all-floating FETI methods for the simulation of arterial tissues

High-resolution and anatomically realistic computer models of biological soft tissues play a significant role in the understanding of the function of cardiovascular components in health and disease. However, the computational effort to handle fine grids to resolve the geometries as well as sophisticated tissue models is very challenging. One possibility to derive a strongly scalable parallel solution algorithm is to consider finite element tearing and interconnecting (FETI) methods. In this study we propose and investigate the application of FETI methods to simulate the elastic behavior of biological soft tissues. As one particular example we choose the artery which is - as most other biological tissues - characterized by anisotropic and nonlinear material properties. We compare two specific approaches of FETI methods, classical and all-floating, and investigate the numerical behavior of different preconditioning techniques. In comparison to classical FETI, the all-floating approach has not only advantages concerning the implementation but in many cases also concerning the convergence of the global iterative solution method. This behavior is illustrated with numerical examples. We present results of linear elastic simulations to show convergence rates, as expected from the theory, and results from the more sophisticated nonlinear case where we apply a well-known anisotropic model to the realistic geometry of an artery. Although the FETI methods have a great applicability on artery simulations we will also discuss some limitations concerning the dependence on material parameters.Comment: 29 page

Roll convection of binary fluid mixtures in porous media

We investigate theoretically the nonlinear state of ideal straight rolls in the Rayleigh-B\'enard system of a fluid layer heated from below with a porous medium using a Galerkin method. Applying the Oberbeck-Boussinesq approximation, binary mixtures with positive separation ratio are studied and compared to one-component fluids. Our results for the structural properties of roll convection resemble qualitatively the situation in the Rayleigh--B\'enard system without porous medium except for the fact that the streamlines of binary mixtures are deformed in the so-called Soret regime. The deformation of the streamlines is explained by means of the Darcy equation which is used to describe the transport of momentum. In addition to the properties of the rolls, their stability against arbitrary infinitesimal perturbations is investigated. We compute stability balloons for the pure fluid case as well as for a wide parameter range of Lewis numbers and separation ratios which are typical for binary gas and fluid mixtures. The stability regions of rolls are found to be restricted by a crossroll, a zigzag and a new type of oscillatory instability mechanism, which can be related to the crossroll mechanism

Discovery and Metabolic Engineering of Steroid Alkaloid Biosynthetic Genes from (\u3ci\u3eVeratrum californicum\u3c/i\u3e)

The steroid alkaloid cyclopamine has shown much promise as a treatment for cancers in which aberrant hedgehog signaling plays a role. The compound, originally discovered due to its teratogenic effects in sheep, binds to the hedgehog signaling receptor Smoothend and prevents downstream activation. As this pathway is primarily active during embryonic development, overactivation later in life can lead to tumor formation and proliferation. Several studies have shown that cyclopamine can inhibit and even reverse tumor growth, but limited supply will prevent widespread use upon FDA approval of it or any of its semi-synthetic analogs. As high value plant medicinal compounds are in demand, producing them at industrially feasible levels is not always possible. Many plant natural products are originally produced in low quantities and often from species that are not amendable to cultivation. Production of these compounds in a heterologous system is ideal, but first the underlying genes that encode biosynthetic enzymes must be discovered. Cyclopamine is produced in one of these non-ideal sources, the slow growing Veratrum californicum, a species that has not been successfully cultivated. Our goal was to discover the underlying genes in the biosynthetic pathway to cyclopamine and express them in the emerging heterologous production system Camelina sativa. Herein, the first four genes in the biosynthetic steroid alkaloid pathway to verazine, a hypothesized intermediate of cyclopamine, have been discovered by correlating gene expression and alkaloid accumulation. In addition, the genes have been successfully introduced into C. sativa under the control of seed specific promoters for the production of V. californicum secondary metabolites in seed. Future work requires discovery of the remaining cyclopamine biosynthetic genes and metabolic fine tuning for increased metabolite yield, but the groundwork has been set for future work using C. sativa as a production system for high value, medicinally significant, plant natural products

Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging

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.Comment: This work was supported by Erwin Schroedinger Fellowship J3787-B21 from the Austrian Science Fund and by National Institutes of Health (NIH) R01 Research Grant GM21841. Additionally, this project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Action H2020-MSCA-IF-2016 InsiliCardio, GA No. 75083

Restriction on the energy and luminosity of e+e- storage rings due to beamstrahlung

The role of beamstrahlung in high-energy e+e- storage-ring colliders (SRCs) is examined. Particle loss due to the emission of single energetic beamstrahlung photons is shown to impose a fundamental limit on SRC luminosities at energies 2E_0 >~ 140 GeV for head-on collisions and 2E_0 >~ 40 GeV for crab-waist collisions. With beamstrahlung taken into account, we explore the viability of SRCs in the E_0=240-500 GeV range, which is of interest in the precision study of the Higgs boson. At 2E_0=240 GeV, SRCs are found to be competitive with linear colliders; however, at 2E_0=400-500 GeV, the attainable SRC luminosity would be a factor 15-25 smaller than desired.Comment: Latex, 5 pages. v2 differs only by minor changes is abstract and introduction, one reference is added. v3 corresponds to the paper published in PR