TY - JOUR A1 - Pauli, Martin A1 - Paul, Mila M. A1 - Proppert, Sven A1 - Mrestani, Achmed A1 - Sharifi, Marzieh A1 - Repp, Felix A1 - Kürzinger, Lydia A1 - Kollmannsberger, Philip A1 - Sauer, Markus A1 - Heckmann, Manfred A1 - Sirén, Anna-Leena T1 - Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections JF - Communications Biology N2 - Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution. KW - mossy fiber synapses KW - CA3 pyrimidal cells KW - CA2+ channels KW - active zone KW - hippocampal KW - release KW - plasticity KW - proteins KW - platform KW - reveals Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259830 VL - 4 ER - TY - JOUR A1 - Leal, Andrea Zurita A1 - Schwebs, Marie A1 - Briggs, Emma A1 - Weisert, Nadine A1 - Reis, Helena A1 - Lemgruber, Leondro A1 - Luko, Katarina A1 - Wilkes, Jonathan A1 - Butter, Falk A1 - McCulloch, Richard A1 - Janzen, Christian J. T1 - Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation JF - Nucleic Acids Research N2 - Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to off-spring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation. KW - cross-link repair KW - cell cycle KW - gene expression KW - low fidelity KW - replication KW - bypass KW - theta KW - reveals KW - binding Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230579 VL - 48 IS - 17 ER - TY - JOUR A1 - Niewalda, Thomas A1 - Völler, Thomas A1 - Eschbach, Claire A1 - Ehmer, Julia A1 - Wen-Chuang, Chou A1 - Timme, Marc A1 - Fiala, André A1 - Gerber, Bertram T1 - A Combined Perceptual, Physico-Chemical, and Imaging Approach to 'Odour-Distances' Suggests a Categorizing Function of the Drosophila Antennal Lobe JF - PLoS One N2 - How do physico-chemical stimulus features, perception, and physiology relate? Given the multi-layered and parallel architecture of brains, the question specifically is where physiological activity patterns correspond to stimulus features and/or perception. Perceived distances between six odour pairs are defined behaviourally from four independent odour recognition tasks. We find that, in register with the physico-chemical distances of these odours, perceived distances for 3octanol and n-amylacetate are consistently smallest in all four tasks, while the other five odour pairs are about equally distinct. Optical imaging in the antennal lobe, using a calcium sensor transgenically expressed in only first-order sensory or only second-order olfactory projection neurons, reveals that 3-octanol and n-amylacetate are distinctly represented in sensory neurons, but appear merged in projection neurons. These results may suggest that within-antennal lobe processing funnels sensory signals into behaviourally meaningful categories, in register with the physico-chemical relatedness of the odours. KW - organization KW - cameleon KW - honeybee KW - map KW - neurons KW - reveals KW - melanogaster KW - mushroom body KW - spatial representation KW - olfactory information Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133510 VL - 6 IS - 9 ER - TY - JOUR A1 - Williams, Richard D. A1 - Chagtai, Tasnim A1 - Alcaide-German, Marisa A1 - Apps, John A1 - Wegert, Jenny A1 - Popov, Sergey A1 - Vujanic, Gordan A1 - Van Tinteren, Harm A1 - Van den Heuvel-Eibrink, Marry M A1 - Kool, Marcel A1 - De Kraker, Jan A1 - Gisselsson, David A1 - Graf, Norbert A1 - Gessler, Manfred A1 - Pritchard-Jones, Kathy T1 - Multiple mechanisms of MYCN dysregulation in Wilms tumour JF - Oncotarget N2 - Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation. KW - integrative genomics viewer KW - oncogene amplification KW - sequencing data KW - gene KW - gain KW - copy number KW - somatic mutations KW - beta-catenin KW - histology KW - reveals KW - Wilms tumour KW - MYCN KW - DNA methylation KW - prognostic marker Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143471 VL - 6 IS - 9 ER - TY - JOUR A1 - Burgsdorf, Ilia A1 - Slaby, Beate M. A1 - Handley, Kim M. A1 - Haber, Markus A1 - Blom, Jochen A1 - Marshall, Christopher W. A1 - Gilbert, Jack A. A1 - Hentschel, Ute A1 - Steindler, Laura T1 - Lifestyle Evolution in Cyanobacterial Symbionts of Sponges JF - mBio N2 - The "Candidatus Synechococcus spongiarum" group includes different clades of cyanobacteria with high 16S rRNA sequence identity (~99%) and is the most abundant and widespread cyanobacterial symbiont of marine sponges. The first draft genome of a "Ca. Synechococcus spongiarum" group member was recently published, providing evidence of genome reduction by loss of genes involved in several nonessential functions. However, "Ca. Synechococcus spongiarum" includes a variety of clades that may differ widely in genomic repertoire and consequently in physiology and symbiotic function. Here, we present three additional draft genomes of "Ca. Synechococcus spongiarum," each from a different clade. By comparing all four symbiont genomes to those of free-living cyanobacteria, we revealed general adaptations to life inside sponges and specific adaptations of each phylotype. Symbiont genomes shared about half of their total number of coding genes. Common traits of "Ca. Synechococcus spongiarum" members were a high abundance of DNA modification and recombination genes and a reduction in genes involved in inorganic ion transport and metabolism, cell wall biogenesis, and signal transduction mechanisms. Moreover, these symbionts were characterized by a reduced number of antioxidant enzymes and low-weight peptides of photosystem II compared to their free-living relatives. Variability within the "Ca. Synechococcus spongiarum" group was mostly related to immune system features, potential for siderophore-mediated iron transport, and dependency on methionine from external sources. The common absence of genes involved in synthesis of residues, typical of the O antigen of free-living Synechococcus species, suggests a novel mechanism utilized by these symbionts to avoid sponge predation and phage attack. IMPORTANCE While the Synechococcus/Prochlorococcus-type cyanobacteria are widely distributed in the world's oceans, a subgroup has established its niche within marine sponge tissues. Recently, the first genome of sponge-associated cyanobacteria, " Candidatus Synechococcus spongiarum," was described. The sequencing of three representatives of different clades within this cyanobacterial group has enabled us to investigate intraspecies diversity, as well as to give a more comprehensive understanding of the common symbiotic features that adapt "Ca. Synechococcus spongiarum" to its life within the sponge host. KW - marine Synechococcus strains KW - ribosomal RNA genes KW - single cell KW - vertical transmission KW - microbial communities KW - sequence analysis KW - spacer sequences KW - genomic analysis KW - web server KW - reveals Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143117 VL - 6 IS - 3 ER - TY - JOUR A1 - Litovkin, Kirill A1 - Van Eynde, Aleyde A1 - Joniau, Steven A1 - Lerut, Evelyne A1 - Laenen, Annouschka A1 - Gevaert, Thomas A1 - Gevaert, Olivier A1 - Spahn, Martin A1 - Kneitz, Burkhard A1 - Gramme, Pierre A1 - Helleputte, Thibault A1 - Isebaert, Sofie A1 - Haustermans, Karin A1 - Bollen, Mathieu T1 - DNA Methylation-Guided Prediction of Clinical Failure in High-Risk Prostate Cancer JF - PLoS ONE N2 - Background Prostate cancer (PCa) is a very heterogeneous disease with respect to clinical outcome. This study explored differential DNA methylation in a priori selected genes to diagnose PCa and predict clinical failure (CF) in high-risk patients. Methods A quantitative multiplex, methylation-specific PCR assay was developed to assess promoter methylation of the APC, CCND2, GSTP1, PTGS2 and RARB genes in formalin-fixed, paraffin-embedded tissue samples from 42 patients with benign prostatic hyperplasia and radical prostatectomy specimens of patients with high-risk PCa, encompassing training and validation cohorts of 147 and 71 patients, respectively. Log-rank tests, univariate and multivariate Cox models were used to investigate the prognostic value of the DNA methylation. Results Hypermethylation of APC, CCND2, GSTP1, PTGS2 and RARB was highly cancer-specific. However, only GSTP1 methylation was significantly associated with CF in both independent high-risk PCa cohorts. Importantly, trichotomization into low, moderate and high GSTP1 methylation level subgroups was highly predictive for CF. Patients with either a low or high GSTP1 methylation level, as compared to the moderate methylation groups, were at a higher risk for CF in both the training (Hazard ratio [HR], 3.65; 95% CI, 1.65 to 8.07) and validation sets (HR, 4.27; 95% CI, 1.03 to 17.72) as well as in the combined cohort ( HR, 2.74; 95% CI, 1.42 to 5.27) in multivariate analysis. Conclusions Classification of primary high-risk tumors into three subtypes based on DNA methylation can be combined with clinico-pathological parameters for a more informative risk-stratification of these PCa patients. KW - CpG island hypermethylation KW - radical prostatectomy KW - promoter methylation KW - receptor beta KW - gene KW - GSTP1 KW - biomarkers KW - diagnosis KW - recurrence KW - reveals Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151705 VL - 10 IS - 6 ER - TY - JOUR A1 - Groeneweg, Femke L. A1 - van Royen, Martin E. A1 - Fenz, Susanne A1 - Keizer, Veer I. P. A1 - Geverts, Bart A1 - Prins, Jurrien A1 - de Kloet, E. Ron A1 - Houtsmuller, Adriaan B. A1 - Schmidt, Thomas S. A1 - Schaaf, Marcel J. M. T1 - Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP JF - PLOS ONE N2 - Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (similar to 0.7 s) and the other half for longer time periods (similar to 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (<= 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences. KW - NF-KAPPA-B KW - image correlation spectroscopy KW - human mineralocorticoid receptor KW - nuclear-pore complexes KW - in-vivo KW - living cells KW - mobility KW - transcription KW - protein KW - reveals Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117085 VL - 9 IS - 3 ER - TY - JOUR A1 - Bischler, Thorsten A1 - Kopf, Matthias A1 - Voss, Bjoern T1 - Transcript mapping based on dRNA-seq data JF - BMC Bioinformatics N2 - Background: RNA-seq and its variant differential RNA-seq (dRNA-seq) are today routine methods for transcriptome analysis in bacteria. While expression profiling and transcriptional start site prediction are standard tasks today, the problem of identifying transcriptional units in a genome-wide fashion is still not solved for prokaryotic systems. Results: We present RNASEG, an algorithm for the prediction of transcriptional units based on dRNA-seq data. A key feature of the algorithm is that, based on the data, it distinguishes between transcribed and un-transcribed genomic segments. Furthermore, the program provides many different predictions in a single run, which can be used to infer the significance of transcriptional units in a consensus procedure. We show the performance of our method based on a well-studied dRNA-seq data set for Helicobacter pylori. Conclusions: With our algorithm it is possible to identify operons and 5'- and 3'-UTRs in an automated fashion. This alleviates the need for labour intensive manual inspection and enables large-scale studies in the area of comparative transcriptomics. KW - transcriptional start site KW - dynamic programming KW - RNA-seq KW - differential KW - segmentation KW - transcriptional uni KW - transcriptome KW - reveals KW - model Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116663 SN - 1471-2105 VL - 15 IS - 122 ER - TY - JOUR A1 - Ioakeimidis, Fotis A1 - Ott, Christine A1 - Kozjak-Pavlovic, Vera A1 - Violitzi, Foteini A1 - Rinotas, Vagelis A1 - Makrinou, Eleni A1 - Eliopoulos, Elias A1 - Fasseas, Costas A1 - Kollias, George A1 - Douni, Eleni T1 - A Splicing Mutation in the Novel Mitochondrial Protein DNAJC11 Causes Motor Neuron Pathology Associated with Cristae Disorganization, and Lymphoid Abnormalities in Mice JF - PLOS ONE N2 - Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases. KW - dominant optic atrophy KW - amyotrophic-lateral-sclerosis KW - nervous system KW - membrane organization KW - mitofilin KW - data-bank KW - model KW - biogenesis KW - morphology KW - reveals Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115581 VL - 9 IS - 8 ER -