Mig1 localization exhibits biphasic behavior which is controlled by both metabolic and regulatory roles of the sugar kinases

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeastSaccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role

Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca

Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca ( Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested

Scalable and flexible inference framework for stochastic dynamic single-cell models

Understanding the inherited nature of how biological processes dynamically change over time and exhibit intra- and inter-individual variability, due to the different responses to environmental stimuli and when interacting with other processes, has been a major focus of systems biology. The rise of single-cell fluorescent microscopy has enabled the study of those phenomena. The analysis of single-cell data with mechanistic models offers an invaluable tool to describe dynamic cellular processes and to rationalise cell-to-cell variability within the population. However, extracting mechanistic information from single-cell data has proven difficult. This requires statistical methods to infer unknown model parameters from dynamic, multi-individual data accounting for heterogeneity caused by both intrinsic (e.g. variations in chemical reactions) and extrinsic (e.g. variability in protein concentrations) noise. Although several inference methods exist, the availability of efficient, general and accessible methods that facilitate modelling of single-cell data, remains lacking. Here we present a scalable and flexible framework for Bayesian inference in state-space mixed-effects single-cell models with stochastic dynamic. Our approach infers model parameters when intrinsic noise is modelled by either exact or approximate stochastic simulators, and when extrinsic noise is modelled by either time-varying, or time-constant parameters that vary between cells. We demonstrate the relevance of our approach by studying how cell-to-cell variation in carbon source utilisation affects heterogeneity in the budding yeast Saccharomyces cerevisiae SNF1 nutrient sensing pathway. We identify hexokinase activity as a source of extrinsic noise and deduce that sugar availability dictates cell-to-cell variability

An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells

Dynamic heterogeneities in the out-of-equilibrium dynamics of simple spherical spin models

The response of spherical two-spin interaction models, the spherical ferromagnet (s-FM) and the spherical Sherrington-Kirkpatrick (s-SK) model, is calculated for the protocol of the so-called nonresonant hole burning experiment (NHB) for temperatures below the respective critical temperatures. It is shown that it is possible to select dynamic features in the out-of-equilibrium dynamics of both models, one of the hallmarks of dynamic heterogeneities. The behavior of the s-SK model and the s-FM in three dimensions is very similar, showing dynamic heterogeneities in the long time behavior, i.e. in the aging regime. The appearence of dynamic heterogeneities in the s-SK model explicitly demonstrates that these are not necessarily related to {\it spatial} heterogeneities. For the s-FM it is shown that the nature of the dynamic heterogeneities changes as a function of dimensionality. With incresing dimension the frequency selectivity of the NHB diminishes and the dynamics in the mean-field limit of the s-FM model becomes homogeneous.Comment: 16 pages, 8 figure

Photoproduction of meson pairs: First measurement of the polarization observable I^s

The polarization observable I^s, a feature exclusive to the acoplanar kinematics of multi-meson final states produced via linearly polarized photons, has been measured for the first time. Results for the reaction g p -> p pi0 eta are presented for incoming photon energies between 970 MeV and 1650 MeV along with the beam asymmetry I^c. The comparably large asymmetries demonstrate a high sensitivity of I^s to the dynamics of the reaction. Fits using Bonn-Gatchina partial wave analysis demonstrate that the new polarization observables carry significant information on the contributing partial waves.Comment: 11 pages, 6 figures, v2 to appear in Phys. Lett.

Measurement of the Beam Asymmetry Σ\Sigma in the Forward Direction for pi0 Photoproduction

Photoproduction of neutral pions has been studied with the CBELSA/TAPS detector for photon energies between 0.92 and 1.68~GeV at the electron accelerator ELSA. The beam asymmetry~$\Sigma$ has been extracted for $115^\circ < \theta_{\rm c.m.} < 155^\circ$ of the $\pi^0$~meson and for $\theta_{\rm c.m.} < 60^\circ$. The new beam asymmetry data improve the world database for photon energies above 1.5~GeV and, by covering the very forward region, extend previously published data for the same reaction by our collaboration. The angular dependence of $\Sigma$ shows overall good agreement with the SAID parameterization.Comment: 11 pages, 10 figures, published in Phys. Rev. C, included LEPS data and MAID 2007 predictions for comparison with our dat

Photoproduction of Neutral Pions off Protons

Photoproduction of neutral pions has been studied with the CBELSA/TAPS detector in the reaction $\gamma p\to p\pi^0$ for photon energies between 0.85 and 2.50 GeV. The $\pi^0$ mesons are observed in their dominant neutral decay mode: $\pi^0\to\gamma\gamma$. For the first time, the differential cross sections cover the very forward region, $\theta_{\rm c.m.}<60^\circ$. A partial-wave analysis of these data within the Bonn-Gatchina framework observes the high-mass resonances $G_{17}$(2190), $D_{13}$(2080), and $D_{15}$(2070).Comment: 12 pages, 9 figures, minor modifications to PWA sectio

Photoproduction of {\omega} Mesons off the Proton

The differential cross sections and unpolarized spin-density matrix elements for the reaction $\gamma p\to p\omega$ were measured using the CBELSA/TAPS experiment for initial photon energies ranging from the reaction threshold to 2.5 GeV. These observables were measured from the radiative decay of the $\omega$ meson, $\omega\to\pi^0\gamma$. The cross sections cover the full angular range and show the full extent of the $t$-channel forward rise. The overall shape of the angular distributions in the differential cross sections and unpolarized spin-density matrix elements are in fair agreement with previous data. In addition, for the first time, a beam of linearly-polarized tagged photons in the energy range from 1150 MeV to 1650 MeV was used to extract polarized spin-density matrix elements. These data were included in the Bonn-Gatchina partial wave analysis (PWA). The dominant contribution to $\omega$ photoproduction near threshold was found to be the $3/2^+$ partial wave, which is primarily due to the sub-threshold $N(1720)\,3/2^+$ resonance. At higher energies, pomeron-exchange was found to dominate whereas $\pi$-exchange remained small. These $t$-channel contributions as well as further contributions from nucleon resonances were necessary to describe the entire dataset: the $1/2^-$, $3/2^-$, and $5/2^+$ partial waves were also found to contribute significantly.Comment: 8 pages, 5 figures, accepted for publication in Physics Letters