Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress

Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacyl-ends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3′-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.</p

A systematic characterization of microglia-like cell occurrence during retinal organoid differentiation

Cerebral organoids differentiated from human-induced pluripotent stem cells (hiPSC) provide a unique opportunity to investigate brain development. However, organoids usually lack microglia, brain-resident immune cells, which are present in the early embryonic brain and participate in neuronal circuit development. Here, we find IBA1+ microglia-like cells alongside retinal cups between week 3 and 4 in 2.5D culture with an unguided retinal organoid differentiation protocol. Microglia do not infiltrate the neuroectoderm and instead enrich within non-pigmented, 3D-cystic compartments that develop in parallel to the 3D-retinal organoids. When we guide the retinal organoid differentiation with low-dosed BMP4, we prevent cup development and enhance microglia and 3D-cysts formation. Mass spectrometry identifies these 3D-cysts to express mesenchymal and epithelial markers. We confirmed this microglia-preferred environment also within the unguided protocol, providing insight into microglial behavior and migration and offer a model to study how they enter and distribute within the human brain

Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation

In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln, Zo-1, and Cldn4, the proliferation marker Ki67, and the proinflammatory cytokine Tnfα. Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development

Unusual, Virulence Plasmid-Dependent Growth Behavior of Yersinia enterocolitica in Three-Dimensional Collagen Gels▿

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37°C. Motility at 37°C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVΔyadA-carrying yersiniae formed loosely packed microcolonies at 37°C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca2+-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions

Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress.

Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacyl-ends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3'-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs

Human CCA-adding enzyme is able to repair damaged CCA ends of tRNAs.

<p>(A) Total HeLa tRNAs and (B) internally radioactively labeled yeast tRNA^Phe were incubated successively with angiogenin and human CCA-adding enzyme. Subsequently to the angiogenin treatment (4 h), T4 polynucleotide kinase (PNK) (45 min) was added which converts the 2′,3′-cyclophosphate ends <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003767#pgen.1003767-Rybak1" target="_blank">[42]</a> generated by the angiogenin cleavage to free 3′OH. After purification tRNAs were subjected to treatment with the CCA-adding enzyme (30 min). The 3′-CCA end integrity of the HeLa tRNAs was determined with the fluorescent oligonucleotide (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003767#pgen-1003767-g001" target="_blank">Figure 1A</a>, schematic inset). tRNA^Phe lacking the terminal 3′-adenosine (tRNA^PheCC) served as a control. The numbers on the left denote the DNA ladder in nt.</p

Scanning-independent translation initiation is less influenced by low dose oxidative stress.

<p>(A) Polysomal profiles of untreated or arsenite treated HeLa cells. (B) Schematic of the plasmid used to monitor cap-dependent and IRES-dependent translation initiation. Inhibition of cap-dependent, Rluc (C) or IRES-mediated, Fluc (D) translation upon exposure to oxidative stress. HeLa cells expressing the bicistronic construct encoding Rluc under the CMV-promoter (scanning-dependent translation) and Fluc under the CrPV-IRES (non-scanning controlled translation) were exposed to different arsenite concentrations for various times. Addition of DMSO to the cells served as a control. Data in (C) and (D) ± SEM are normalized to the first data point for which the activity was set as 100.</p

Oxidative stress-mediated tRNA cleavage in HeLa cells.

<p>(A) Minor fraction tiRNAs are generated by longer exposure to 500 µM arsenite (>30 min). The numbers on the left denote the DNA ladder in nt. (B) Arsenite alters the integrity of the 3′-CCA end of full-length tRNAs in a dose-dependent manner. The amount of tRNAs with intact CCA ends was analyzed by their ability to ligate to a fluorescently labeled oligonucleotide (schematic inset) which forms a loop and binds only intact 3′-CCA ends (upper two gels). The intensity of tRNAs with intact CCA termini was quantified from the gels, normalized to the total tRNA amount at each time point and presented as relative values ± SEM (from three independent experiments) to the amount of initial, untreated sample which was set as 1.0. The amount of the total tRNA remained almost unchanged when cells were exposed to arsenite (500 µM) and visualized by SYBR Green (bottom gel marked as total tRNA). (C) Increase of the cellular concentration of angiogenin alters the 3′-CCA integrity of tRNAs. Angiogenin was upregulated by ectopic expression under the control of a CMV promoter for 8 h (+angiogenin). The sample representing arsenite stress (+arsenite) corresponds to the 60-min data point at 500 µM arsenite in panel (B) and is used for comparison. The intensity of tRNAs with intact CCA termini was the quantified as described for panel (B). ** for <i>p</i><0.01.</p

Uncovering brain tissue architecture across scales with super-resolution light microscopy

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the system’s molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons