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Authors: Szczurek, Aleksander
Title: Single molecule localisation microscopy by photoconversion and dynamic labelling using DNA-binding dyes resolves chromatin compaction in model ischemia
Online publication date: 17-Mar-2017
Language: english
Abstract: Single molecule localisation microscopy (SMLM) is a method based on switching and subsequent nanometre-precision localisation of isolated fluorophore signals for super-resolution fluorescence microscopy. Thus far, this method has been successfully applied to imaging of various cellular structures including e.g. cytoskeleton, mitochondria, neuronal axons and many others, revealing manifold previously unknown cellulardetails. Strikingly, SMLM was applied to the DNA study only to a minor degree. Whereas a higher order DNA (chromatin) organisation in the cell nucleus still remainspuzzling, previous studies using SMLM focusedrather on imaging of isolated DNA molecules in vitro. In this work novel solutions for super-resolution DNA imaging in eukaryotic cell nuclei were developed utilising SMLM principles. First approach relies on UV-induced photoconversion of DNA-binding Hoechst and DAPI dyes. By applying low intensity near-UV light a stochastic photoconversion of DNA-bound dyes occurs and single molecule signals can be discriminated based on spectral properties. Second approach utilises a previously developed dynamic DNA labelling (Binding Activated Localisation Microscopy) with directly binding dyes that upon transient association with the DNA become fluorescent. In turn, while dynamically dissociated are non-fluorescent. In this study different aspects of DNA chemistry andDNA-binding werestudied to enhance understanding of BALM mechanism and to facilitate applicationextensionto the nuclei of mammals and rodents. Both methods led to 3 – 5 fold improvement in resolution as compared to a conventional fluorescence imaging. Chromatin, a complex of DNA and proteins, is known to directly respond and adapt to changes in environmental conditions just as temperature, ionic strength and many others. Currently among diseases involving such environmental balance disruptions, ischemia, a blood insufficiency,is1st(cardiovascular disease) and 2nd(stroke) cause of death worldwide according to the World Health Organisation.Knowing that epigenetic landscape in ischemia undergoes a massive remodelling e.g. involving a general deacetylation of chromatin, we anticipated that chromatin structure undergoes a change. We tested this hypothesis using aforementioned superresolution microscopy methodology. This approach revealed previously unknown chromatin condensation towards a nuclear periphery. In support we found that ischemic chromatin bore a decreased DNase Idigestion susceptibility and decreased linker histone H1dynamics. We hypothesise that thesource of chromatin condensation are polyamines and divalent cations otherwise complexed with ATP. Upon ATP depletion in ischemia they would relocate to the cell nucleus and bind to negatively charged DNA inducing its compaction. The results presented in this dissertation have an impact on better understanding and interpretation of previous studies on ischemiaand may help to design a future therapy based on a novel aspect. The super-resolution methodologymay prove useful for studying effects of other environmental factorse.g. oxidative stress or to investigate chromatin texture in cancerous cells to assess their malignancy.
DDC: 570 Biowissenschaften
570 Life sciences
Institution: Johannes Gutenberg-Universität Mainz
Department: Externe Einrichtungen
Place: Mainz
Version: Original work
Publication type: Dissertation
License: in Copyright
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Extent: 97 Seiten
Appears in collections:JGU-Publikationen

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