1,720,976 research outputs found
Post-Translational Modifications Soften Intermediate Filaments
No full textThe mechanical properties of biological cells are determined by the cytoskeleton, a composite biopolymer network consisting of microtubules, actin filaments and intermediate filaments (IFs). By differential expression of the cytoskeletal proteins, modulation of the network architecture and the interactions between the filaments, cell mechanics may be adapted to varying requirements on the cell. Here, we focus on the intermediate filament protein vimentin and introduce post-translational modifications as an additional, much faster mechanism for mechanical modulation. We study the impact of phosphorylation on filament mechanics by recording precise force-strain curves using optical traps. Whereas full phosphorylation leads to disassembly of IFs, partial phosphorylation results in softening of the filaments. We show that binding of the protein 14–3–3 to phosphorylated vimentin IFs further enhances this effect and speculate that in the cell 14–3–3 may serve to preserve the softening and thereby the altered cell mechanics. By employing phosphomimetic mutants and complementary Monte Carlo simulations, we explain our observation through the additional charges introduced during
Ca2+-triggered (de)ubiquitination events in synapses
No full text501100001659 German Research Foundatio
Relative Quantification of Phosphorylated and Glycosylated from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy
No full textExtracellular vesicles derived from the choroid plexus trigger the differentiation of neural stem cells
Full text linkThe choroid plexus secrets cerebrospinal fluid (CSF) composed of electrolytes, cytokines, growth factors, metabolites and extracellular vesicles (EVs) that flow through the interconnected brain ventricles. On their course, CSF components can act as signals that affect, for example, neural stem cells (NSCs) residing in niches of the ventricular wall. We studied EV‐born CSF signals in an in vitro culture system. We purified EVs from the secretome of a choroid plexus cell line (Z310 cells), and from primary choroid plexus cultures and co‐cultured those EVs with NSCs isolated from the niche of the lateral and the third ventricle. EVs(Z310) and EVs(CHP) were purified by differential centrifugation. This yielded fractions of EVs of 50–150‐nm diameter that induced a complex multicellular network formation and NSC differentiation. Both types of EV converted the round NSCs to cells that extended long processes that contacted nearby, alike‐shaped cells. Mass spectrometry showed that the differentiation‐inducing EV(Z310) were enriched for membrane and membrane‐associated proteins involved in cell differentiation, membrane trafficking, and membrane organization. We hypothesize that this type of EV (Z310) cargo causes changes of stem cell morphology that leads to multicellular networks in the niches. This cell‐shape transition may represent an initial step in NSC differentiation
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Evaluation and optimization of high-field asymmetric waveform ion-mobility spectrometry for multiplexed quantitative site-specific N-glycoproteomics
No full textThe heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics
Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles
Full text linkNeurotransmitter release is a key step that enables information flow between the pre- and
post-synapse. However, regulation of the neurotransmitter release remains an intricate and
widely unexplored matter despite recent advances in the understanding of the
neurotransmitter release machinery and the analysis of the synaptic proteome and protein
modifications. Indeed, post-translational protein modifications such as phosphorylation are
suitable to quickly fine-tune the neurotransmitter release “in place” via affecting tertiary protein
structures and protein-protein interactions, and globally, via modulating signaling pathways.
Here, the investigations were focused on the dependence of protein phosphorylation in
synaptosomes on the synaptic vesicle (SV) cycling, determining kinase-substrate interactions,
and modulatory effects of selected sites on exo- and endocytosis.
The analysis of synaptic phosphoproteome was conducted using TiO2-based enrichment of
phosphorylated peptides with subsequent chemical labeling by isobaric mass tags (TMT) and
a mass spectrometry-based quantification. Synaptosomes were employed as a functional
model of a synapse as they contain the required neurotransmitter release machinery and
respond to stimulation. First, the applicability of electrical stimulation was tested. The field-
stimulation evoked reproducible glutamate release that was significantly suppressed in the
absence of Ca2+, though it remained uncertain, to which degree the release is governed by
exocytosis. Therefore, another approach using a KCl-induced depolarization and treatment
with botulinum neurotoxins (BoNTs) was used to identify phosphorylation events that depend
on SV cycling. BoNTs cleave specifically SNARE proteins and thus block exocytosis and SV
cycling, but do not impede Ca2+-influx evoked by the plasma membrane depolarization.
Comparison of phosphorylation events in synaptosomes stimulated in the presence of Ca2+,
EGTA (0 net Ca2+) or pre-treated with BoNTs identified sites that were differentially
phosphorylated following BoNT treatment, i.e., SV-cycling-dependent sites, and sites that
were differentially phosphorylated when comparing Ca and EGTA conditions, but did not
change under BoNT treatment, i.e., primarily Ca2+-dependent sites. Further differential
expression analysis revealed that BoNT-treatment mostly caused de-phosphorylation of
synaptic proteins. A kinase-substrate analysis showed that >25% of BoNT-responsive sites
are predicted MAPK substrates and 20% of
primarily Ca2+-dependent sites are presumably regulated by CaMKII, which corroborates Ca2+-
dependence of these phosphorylation events. SV-cycling-dependent phosphorylation sites on
syntaxin-1 (T21/T23-Stx1), synaptobrevin (S75-Vamp2), and cannabinoid receptor-1
(S314/T322-Cnr1) were further investigated for their impact on exo- and endocytosis. In
collaboration with Dr. Eugenio Fornasiero and Prof. Dr. Silvio O. Rizzoli, corresponding
phosphomimetic and non-phosphorylatable variants of the proteins were expressed in
cultured hippocampal neurons. Imaging of the pH-sensor pHluorine coupled to
synaptobrevin-2 revealed that the expression of phosphomimetic and non-phosphorylatable
sites affected exo- and endocytosis in neurons.
This work is first to investigate the electrical stimulation in relation to the Ca2+-dependent
neurotransmitter release and exocytosis in synaptosomes. It further provides a
comprehensive draft of synaptosomal phosphoproteome and is first to demonstrate its global
dependence on an active SV cycling. The analysis of cultured hippocampal neurons
expressing non-phosphorylatable and phosphomimetic mutants of pre-synaptic proteins
syntaxin-1, synaptobrevin-2, and cannabinoid receptor-1 further demonstrates that the
identified SV-cycling-dependent sites affect exo- and endocytosis.2021-11-0
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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