dictyNews
Electronic Edition
Volume 43, number 12
June 9, 2017
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Abstracts
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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 [[log in to unmask]]
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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 [[log in to unmask]]
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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 [[log in to unmask]]
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[End dictyNews, volume 43, number 12]
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