dictyNews Electronic Edition Volume 43, number 12 June 9, 2017 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Hydrophilic interaction anion exchange for separation of multiply-modified neutral and anionic Dictyostelium N-glycans Alba Hykollari, Daniel Malzl, Shi Yan, Iain B. H. Wilson and Katharina Paschinger Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria Electrophoresis, in press DOI: 10.1002/elps.201700073 The unusual nature of the N-glycans of the cellular slime mould Dictyostelium discoideum has been revealed by a number of studies, primarily based on examination of radiolabelled glycopeptides but more recently also by mass spectrometry. The complexity of the N-glycomes of even glycosylation mutants is compounded by the occurrence of anionic modifications, which also present an analytical challenge. In this study, we have employed hydrophilic interaction anion exchange (HIAX) HPLC in combination with MALDI-TOF MS/MS to explore the anionic N-glycome of the M31 (modA) strain, which lacks endoplasmic reticulum alpha-glucosidase II, an enzyme conserved in most eukaryotes including Homo sapiens. Pre-fractionation with HIAX chromatography enabled the identification of N-glycans with unusual oligo-alpha1,2-mannose extensions as well as others with up to four anionic modifications. Due to the use of hydrofluoric acid treatment, we were able to discriminate isobaric glycans differing in the presence of sulphate or phosphate on intersected structures as opposed to those carrying GlcNAc-phosphodiesters. The latter represent biosynthetic intermediates during the pathway leading to formation of the methylphosphorylated mannose epitope, which may have a similar function in intracellular targeting of hydrolases as the mannose-6-phosphate modification of lysosomal enzymes in mammals. In conclusion, HIAX in combination with MS is a highly-sensitive approach for both fine separation and definition of neutral and anionic N-glycan structures.. submitted by: Iain Wilson [iain.wilson@boku.ac.at] ——————————————————————————————————————— Generation of Single-Cell Transcript Variability by Repression Vlatka Antolovic Agnes Miermont, Adam M. Corrigan, Jonathan R. Chubb Laboratory for Molecular Cell Biology and Division of Cell and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK Current Biology, in press Gene expression levels vary greatly within similar cells, even within clonal cell populations [1]. These spontaneous expression differences underlie cell fate diversity in both differentiation and disease [2]. The mechanisms responsible for generating expression variability are poorly understood. Using single-cell transcriptomics, we show that transcript variability emerging during Dictyostelium differentiation is driven predominantly by repression rather than activation. The increased variability of repressed genes was observed over a broad range of expression levels, indicating that variability is actively imposed and not a passive statistical effect of the reduced numbers of molecules accompanying repression. These findings can be explained by a simple model of transcript production, with expression controlled by the frequency, rather than the magnitude, of transcriptional firing events. Our study reveals that the generation of differences between cells can be a direct consequence of the basic mechanisms of transcriptional regulation. submitted by: Jonathan Chubb [j.chubb@ucl.ac.uk] ——————————————————————————————————————— Symmetry breaking in development and stochastic gene expression Jonathan R. Chubb MRC Laboratory for Molecular Cell Biology and Department of Cell and Developmental Biology, University College London, Gower Street, London, WC1E 6BT. WIRES Developmental Biology, in press The prevailing emphasis in developmental biology since the expansion of the molecular biology age has been that developmental decisions are instructive. A cell differentiates to become a specific cell type because it receives a signal, whereas its neighbour, that does not receive the signal, adopts a different fate. This emphasis has been generally accepted, largely because of the success of this view in tractable invertebrate model organisms, and the widespread similarities in molecular regulation to the development of more complex species. An alternative emphasis, that cells make their own decisions, has until the past decade been conspicuously silent. Here I trace the re-emergence of our appreciation of single cell decision-making in development, and how widespread this phenomenon is likely to be. I will focus the discussion on the potential role of stochastic gene expression in generating differences between cells in the absence of simple instructive signals and highlight the complexity of systems proposed to involve this type of regulation. Finally, I will discuss the approaches required to fully test hypotheses that noisy gene regulation can be extrapolated through developmental time to accurately specify cell fate. submitted by: Jonathan Chubb [j.chubb@ucl.ac.uk] ============================================================== [End dictyNews, volume 43, number 12]