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  • Cremer, Christoph  (235)
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  • 1
    Language: English
    In: Human Genetics, 2014, Vol.133(4), pp.403-416
    Description: In line with the intentions of an issue celebrating the 50th anniversary of Human Genetics, we focus on a series of frequently cited studies published in this journal during the 1980s and 1990s. These studies have contributed to the rise of molecular cytogenetics. They yielded evidence that chromosomes occupy distinct territories in the mammalian cell nucleus, first obtained with laser-UV-microbeam experiments and thereafter with chromosome painting, and contributed to the development of interphase cytogenetics and comparative genome hybridization. We provide a personal account of experimental concepts, which were developed by us and others, and describe some of the unforeseeable turns and obstacles, which we had to overcome on the way towards an experimental realization. We conclude with a perspective on current developments and goals of molecular cytogenetics.
    Keywords: Cytogenetics ; Genetic Research ; Chromosomes ; Cells (Biology) ; Genomes ; Genomics;
    ISSN: 0340-6717
    E-ISSN: 1432-1203
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 09 November 2010, Vol.107(45), pp.19426-31
    Description: Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region ("entry site") to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance.
    Keywords: Self Tolerance ; Epigenesis, Genetic -- Immunology ; Epithelial Cells -- Metabolism ; Thymus Gland -- Cytology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: German
    In: Biologie in unserer Zeit, October 2016, Vol.46(5), pp.290-299
    Description: Gespeicherte genetische Information allein nützt nichts, wenn sie nicht zur richtigen Zeit und am richtigen Ort abgerufen werden kann. Die Verpackung der DNA im Chromatin des Zellkerns und dessen dynamische, raum‐zeitliche Anordnung haben einen entscheidenden Einfluss auf die Genexpression und weitere Funktionen des Zellkerns. In dieser Übersichtsarbeit beschreiben die Verfasser die Entwicklung der experimentellen Zellkernarchitektur‐Forschung und die damit einhergehenden Veränderungen der Vorstellungen zur funktionellen Organisation des Zellkerns. Nuclear architecture Stored genetic information is useless, if it cannot be retrieved at the right time and the right place. Packaging of DNA within the chromatin and dynamic changes of its spatiotemporal arrangements of the cell nucleus have a fundamental impact on gene expression and other nuclear functions. In this review the authors describe the development of experimental research on nuclear architecture and of corresponding changes of concepts about the functional organization of the cell nucleus. Zellkerne sind die Informations‐ und Steuerzentrale der Zellen aller eukaryotischen Lebewesen. Neue Erkenntnisse zeigen, dass sie also viel mehr sind als “chemische Reagenzgläser”, in denen komplizierte biochemische Prozesse an Chromatinfäden in wässriger Lösung ablaufen. In ihrer Architektur sind sie mit einer Stadt in der Zelle vergleichbar, deren Funktionen von der räumlichen Anordnung der darin enthaltenen DNA, RNA und Proteine abhängen.
    Keywords: Zellkernarchitektur ; Chromosomenterritorien ; Chromatindomänen ; Laser‐Mikrobestrahlung ; Konfokale Laser‐Scanning Mikroskopie
    ISSN: 0045-205X
    E-ISSN: 1521-415X
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  • 4
    Language: English
    In: Physik in unserer Zeit, 01/2011, Vol.42(1), pp.21-29
    Description: Ein Jahrhundert lang galt die von Ernst Abbe und Lord Rayleigh postulierte Grenze der lichtmikroskopischen Auflösung bei etwa 200 nm als unüberwindlich. Neue physikalische und optoelektronische Verfahren haben sie durchbrochen. Das ihnen zugrunde liegende Prinzip der Lokalisationsmikroskopie nutzt aus, dass näher zusammen liegende Lichtpunkte, etwa zur Fluoreszenz angeregte Moleküle, durch ihre unterschiedlichen spektralen Signaturen räumlich trennbar sind. Solche Signaturen können zum Beispiel verschiedene Fluoreszenzfarben sein oder zeitlich differierende und damit trennbare Fluoreszenzübergänge. Inzwischen sind mehrere Varianten experimentell realisiert. Diese Verfahren können mit sichtbarem Licht Nanostrukturen bis ins molekulare Detail abbilden. Dies gelingt zunehmend auch an lebenden Zellen.
    Keywords: Physics;
    ISSN: Physik in unserer Zeit
    E-ISSN: 00319252
    E-ISSN: 15213943
    Source: Wiley (via CrossRef)
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  • 5
    Language: English
    In: BBA - Biomembranes, April 2014, Vol.1838(4), pp.1191-1198
    Description: In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1 × 10 individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54 nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5–6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.
    Keywords: Superresolution/Localization Microscopy ; Nanoscopy ; Influenza Viruses ; Hepatocyte Growth Factor Receptor ; Single Molecules ; Spdm ; Smlm ; Salm ; Chemistry
    ISSN: 0005-2736
    E-ISSN: 1879-2642
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  • 6
    Language: English
    In: The European Physical Journal H, 2013, Vol.38(3), pp.281-344
    Description: We survey the history of resolution enhancement techniques in microscopy and their impact on current research in biomedicine. Often these techniques are labeled superresolution, or enhanced resolution microscopy, or light-optical nanoscopy. First, we introduce the development of diffraction theory in its relation to enhanced resolution; then we explore the foundations of resolution as expounded by the astronomers and the physicists and describe the conditions for which they apply. Then we elucidate Ernst Abbe’s theory of optical formation in the microscope, and its experimental verification and dissemination to the world wide microscope communities. Second, we describe and compare the early techniques that can enhance the resolution of the microscope. Third, we present the historical development of various techniques that substantially enhance the optical resolution of the light microscope. These enhanced resolution techniques in their modern form constitute an active area of research with seminal applications in biology and medicine. Our historical survey of the field of resolution enhancement uncovers many examples of reinvention, rediscovery, and independent invention and development of similar proposals, concepts, techniques, and instruments. Attribution of credit is therefore confounded by the fact that for understandable reasons authors stress the achievements from their own research groups and sometimes obfuscate their contributions and the prior art of others. In some cases, attribution of credit is also made more complex by the fact that long term developments are difficult to allocate to a specific individual because of the many mutual connections often existing between sometimes fiercely competing, sometimes strongly collaborating groups. Since applications in biology and medicine have been a major driving force in the development of resolution enhancing approaches, we focus on the contribution of enhanced resolution to these fields.
    Keywords: Physicists -- Methods ; Microscopy -- Methods;
    ISSN: 2102-6459
    E-ISSN: 2102-6467
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  • 7
    Language: English
    In: 2012, Vol.7(9), p.e44776
    Description: P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.
    Keywords: Research Article ; Biology ; Physics ; Cell Biology ; Physics ; Biochemistry
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: PLoS ONE, 2012, Vol.7(2), p.e31128
    Description: Tight Junctions (TJ) regulate paracellular permeability of tissue barriers. Claudins (Cld) form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity). To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy. ; TJ-strands were reconstituted by stable transfection of HEK293 cells with the barrier-forming Cld3 or Cld5. Strands were investigated at cell-cell contacts by Spectral Position Determination Microscopy (SPDM), a method of localization microscopy using standard fluorophores. Extended TJ-networks of Cld3-YFP and Cld5-YFP were observed. For each network, 200,000 to 1,100,000 individual molecules were detected with a mean localization accuracy of ∼20 nm, yielding a mean structural resolution of ∼50 nm. Compared to conventional fluorescence microscopy, this strongly improved the visualization of strand networks and enabled quantitative morphometric analysis. Two populations of elliptic meshes (mean diameter 〈100 nm and 300–600 nm, respectively) were revealed. For Cld5 the two populations were more separated than for Cld3. Discrimination of non-polymeric molecules and molecules within polymeric strands was achieved. For both subtypes of claudins the mean density of detected molecules was similar and estimated to be ∼24 times higher within the strands than outside the strands. ; The morphometry and single molecule information provided advances the mechanistic analysis of paracellular barriers. Applying this novel method to different TJ-proteins is expected to significantly improve the understanding of TJ on the molecular level.
    Keywords: Research Article ; Biology ; Computer Science ; Physics ; Computational Biology ; Biophysics ; Computer Science ; Physics ; Biochemistry
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 24 November 2015, Vol.112(47), pp.14635-40
    Description: During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.
    Keywords: Chromatin Organization ; Epigenetics ; Histone Modifications ; Meiosis ; Superresolution Microscopy ; Pachytene Stage ; Chromatin -- Chemistry ; Chromosomes, Mammalian -- Metabolism ; Microscopy -- Methods
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 10
    Language: English
    In: Cancer Research, 08/01/2015, Vol.75(15 Supplement), pp.204-204
    ISSN: 0008-5472
    E-ISSN: 1538-7445
    Source: CrossRef
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