Sancar G.,University of Heidelberg |
Sancar C.,University of Heidelberg |
Brugger B.,University of Heidelberg |
Ha N.,BioQuant Center |
And 8 more authors.
Molecular Cell | Year: 2011
The white-collar complex (WCC), the core transcription factor of the circadian clock of Neurospora, activates morning-specific expression of the transcription repressor CSP1. Newly synthesized CSP1 exists in a transient complex with the corepressor RCM1/RCO1 and the ubiquitin ligase UBR1. CSP1 is rapidly hyperphosphorylated and degraded via UBR1 and its ubiquitin conjugase RAD6. Genes controlled by CSP1 are rhythmically expressed and peak in the evening (i.e., in antiphase to morning-specific genes directly controlled by WCC). Rhythmic expression of these second-tier genes depends crucially on phosphorylation and rapid turnover of CSP1, which ensures tight coupling of CSP1 abundance and function to the circadian activity of WCC. Negative feedback of CSP1 on its own transcription buffers the amplitude of CSP1-dependent oscillations against fluctuations of WCC activity. CSP1 predominantly regulates genes involved in metabolism. It controls ergosterol synthesis and fatty acid desaturases and thereby modulates the lipid composition of membranes. © 2011 Elsevier Inc.
Schneider J.,German Cancer Research Center |
Schneider J.,Bioquant Center |
Zahn J.,German Cancer Research Center |
Zahn J.,Bioquant Center |
And 12 more authors.
Nature Methods | Year: 2015
Electro-optical scanning (>1,000 frames/s) with pixel dwell times on the order of the lifetime of the fluorescent molecular state renders stimulated emission depletion (STED) nanoscopy temporally stochastic. Photon detection from a molecule occurs stochastically in one of several scanning frames, and the spatial origin of the photon is known with subdiffraction precision. Images are built up by binning consecutive frames, making the time resolution freely adjustable. We demonstrated nanoscopy of vesicle motions in living Drosophila larvae and the cellular uptake of viral particles with 5- to 10-ms temporal resolution. © 2015 Nature America, Inc.
Luijsterburg M.S.,University of Amsterdam |
Luijsterburg M.S.,Netherlands Institute for Systems Biology |
Luijsterburg M.S.,Max Delbrück Center for Molecular Medicine |
Von Bornstaedt G.,German Cancer Research Center |
And 13 more authors.
Journal of Cell Biology | Year: 2010
To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes. DNA repair is orchestrated by the interplay of reversible protein-binding events and progressive enzymatic modifications of the chromatin substrate. We demonstrate that faithful recognition of DNA lesions is time consuming, whereas subsequently, repair complexes form rapidly through random and reversible assembly of NER proteins. Our kinetic analysis of the NER system reveals a fundamental conflict between specificity and efficiency of chromatin-associated protein machineries and shows how a trade off is negotiated through reversibility of protein binding. © 2010 Luijsterburg et al.
Mariani L.,German Cancer Research Center |
Mariani L.,Humboldt University of Berlin |
Mariani L.,German Rheumatism Research Center |
Schulz E.G.,German Cancer Research Center |
And 13 more authors.
Molecular Systems Biology | Year: 2010
Although cell-to-cell variability has been recognized as an unavoidable consequence of stochasticity in gene expression, it may also serve a functional role for tuning physiological responses within a cell population. In the immune system, remarkably large variability in the expression of cytokine genes has been observed in homogeneous populations of lymphocytes, but the underlying molecular mechanisms are incompletely understood. Here, we study the interleukin-4 gene (il4) in T-helper lymphocytes, combining mathematical modeling with the experimental quantification of expression variability and critical parameters. We show that a stochastic rate-limiting step upstream of transcription initiation, but acting at the level of an individual allele, controls il4 expression. Only a fraction of cells reaches an active, transcription-competent state in the transient time window determined by antigen stimulation. We support this finding by experimental evidence of a previously unknown short-term memory that was predicted by the model to arise from the long lifetime of the active state. Our analysis shows how a stochastic mechanism acting at the chromatin level can be integrated with transcriptional regulation to quantitatively control cell-to-cell variability. © 2010 EMBO and Macmillan Publishers Limited.
Bingen P.,German Cancer Research Center |
Bingen P.,Bioquant Center |
Reuss M.,German Cancer Research Center |
Reuss M.,Bioquant Center |
And 5 more authors.
Optics Express | Year: 2011
We introduce a parallelized STED microscope featuring m = 4 pairs of scanning excitation and STED beams, providing m-fold increased imaging speed of a given sample area, while maintaining basically all of the advantages of single beam scanning. Requiring only a single laser source and fiber input, the setup is inherently aligned both spatially and temporally. Given enough laser power, the design is readily scalable to higher degrees of parallelization m. © 2011 Optical Society of America.
Brummer A.,German Cancer Research Center |
Brummer A.,BioQuant Center |
Salazar C.,German Cancer Research Center |
Salazar C.,BioQuant Center |
And 4 more authors.
PLoS Computational Biology | Year: 2010
Eukaryotic genomes are duplicated from multiple replication origins exactly once per cell cycle. In Saccharomyces cerevisiae, a complex molecular network has been identified that governs the assembly of the replication machinery. Here we develop a mathematical model that links the dynamics of this network to its performance in terms of rate and coherence of origin activation events, number of activated origins, the resulting distribution of replicon sizes and robustness against DNA rereplication. To parameterize the model, we use measured protein expression data and systematically generate kinetic parameter sets by optimizing the coherence of origin firing. While randomly parameterized networks yield unrealistically slow kinetics of replication initiation, networks with optimized parameters account for the experimentally observed distribution of origin firing times. Efficient inhibition of DNA rereplication emerges as a constraint that limits the rate at which replication can be initiated. In addition to the separation between origin licensing and firing, a time delay between the activation of S phase cyclin-dependent kinase (S-Cdk) and the initiation of DNA replication is required for preventing rereplication. Our analysis suggests that distributive multisite phosphorylation of the S-Cdk targets Sld2 and Sld3 can generate both a robust time delay and contribute to switch-like, coherent activation of replication origins. The proposed catalytic function of the complex formed by Dpb11, Sld3 and Sld2 strongly enhances coherence and robustness of origin firing. The model rationalizes how experimentally observed inefficient replication from fewer origins is caused by premature activation of S-Cdk, while premature activity of the S-Cdk targets Sld2 and Sld3 results in DNA rereplication. Thus the model demonstrates how kinetic deregulation of the molecular network governing DNA replication may result in genomic instability. © 2010 Brümmer et al.
Reuss M.,German Cancer Research Center |
Reuss M.,Bioquant Center |
Engelhardt J.,German Cancer Research Center |
Engelhardt J.,Bioquant Center |
And 3 more authors.
Optics Express | Year: 2010
Stimulated emission depletion (STED) microscopy usually employs a scanning excitation beam that is superimposed by a donut-shaped STED beam for keeping the fluorophores at the periphery of the excitation spot dark. Here, we introduce a simple birefringent device that produces a donut-shaped focal spot with suitable polarization for STED, while leaving the excitation spot virtually intact. The device instantly converts a scanning (confocal) microscope with a co-aligned STED beam into a full-blown STED microscope. The donut can be adapted to reveal, through the resulting fluorescence image, the orientation of fluorophores in the sample, thus directly providing subdiffraction resolution images of molecular orientation. © 2010 Optical Society of America.
Mallm J.-P.,German Cancer Research Center |
Mallm J.-P.,Bioquant Center |
Rippe K.,German Cancer Research Center |
Rippe K.,Bioquant Center
Cell Reports | Year: 2015
Non-coding RNAs can modulate histone modifications that, at the same time, affect transcript expression levels. Here, we dissect such a network in mouse embryonic stem cells (ESCs). It regulates the activity of the reverse transcriptase telomerase, which synthesizes telomeric repeats at the chromosome ends. We find that histone H3 serine 10 phosphorylation set by Aurora kinase B (AURKB) in ESCs during the S phase of the cell cycle at centromeric and (sub)telomeric loci promotes the expression of non-coding minor satellite RNA (. cenRNA). Inhibition of AURKB induces silencing of cenRNA transcription and establishment of a repressive chromatin state with histone H3 lysine 9 trimethylation and heterochromatin protein 1 accumulation. This process results in a continuous shortening of telomeres. We further show that AURKB interacts with both telomerase and cenRNA and activates telomerase in trans. Thus, in mouse ESCs, telomere maintenance is regulated via expression of cenRNA in a cell-cycle-dependent manner. Mallm and Rippe find that AURKB kinase and centromeric RNA regulate telomerase activity. AURKB phosphorylates serine 10 of histone H3 at chromosome p-arms during S phase in embryonic stem cells to induce centromere repeat transcription. Together, AURKB and centromere transcripts activate telomerase and ensure telomere maintenance. © 2015 The Authors.
Hoyer P.,German Cancer Research Center |
Hoyer P.,BioQuant Center |
Staudt T.,German Cancer Research Center |
Staudt T.,BioQuant Center |
And 5 more authors.
Nano Letters | Year: 2011
We demonstrate superresolution fluorescence imaging of cells using bioconjugated CdSe/ZnS quantum dot markers. Fluorescence blueing of quantum dot cores facilitates separation of blinking markers residing closer than the diffraction barrier. The high number of successively emitted photons enables ground state depletion microscopy followed by individual marker return with a resolving power of the size of a single dot (∼12 nm). Nanoscale imaging is feasible with a simple webcam. © 2011 American Chemical Society.
PubMed | BioQuant Center
Type: Journal Article | Journal: Methods of information in medicine | Year: 2016
Coarctation of the aorta is one of the most common congenital heart diseases. Despite different treatment opportunities, long-term outcome after surgical or interventional therapy is diverse. Serial morphologic follow-up of vessel growth is necessary, because vessel growth cannot be predicted by primer morphology or a therapeutic option.For the analysis of the long-term outcome after therapy of congenital diseases such as aortic coarctation, accurate 3D geometric analysis of the aorta from follow-up 3D medical image data such as magnetic resonance angiography (MRA) is important. However, for an objective, fast, and accurate 3D geometric analysis, an automatic approach for 3D segmentation and quantification of the aorta from pediatric images is required.We introduce a new model-based approach for the segmentation of the thoracic aorta and its main branches from follow-up pediatric 3D MRA image data. For robust segmentation of vessels even in difficult cases (e.g., neighboring structures), we propose a new extended parametric cylinder model that requires only relatively few model parameters. Moreover, we include a novel adaptive background-masking scheme used for least-squares model fitting, we use a spatial normalization scheme to align the segmentation results from follow-up examinations, and we determine relevant 3D geometric parameters of the aortic arch.We have evaluated our proposed approach using different 3D synthetic images. Moreover, we have successfully applied the approach to follow-up pediatric 3D MRA image data, we have normalized the 3D segmentation results of follow-up images of individual patients, and we have combined the results of all patients. We also present a quantitative evaluation of our approach for four follow-up 3D MRA images of a patient, which confirms that our approach yields accurate 3D segmentation results. An experimental comparison with two previous approaches demonstrates that our approach yields superior results.From the results, we found that our approach is well suited for the quantification of the 3D geometry of the aortic arch from follow-up pediatric 3D MRA image data. In future work, this will enable to investigate the long-term outcome of different surgical and interventional therapies for aortic coarctation.