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Imaging and Manipulating the Cancer Cell Genome using Light Microscopy

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Light microscopy has played a pivotal role in the study of the architecture of the cell nucleus for more than 150 years. The advent of fluorescence techniques, the striking improvements of temporal and spatial resolution achieved in recent times, and the ability to exploit radiation for targeted interventions ...

Light microscopy has played a pivotal role in the study of the architecture of the cell nucleus for more than 150 years. The advent of fluorescence techniques, the striking improvements of temporal and spatial resolution achieved in recent times, and the ability to exploit radiation for targeted interventions in live cells make modern light microscopy an invaluable tool for unveiling the highly dynamic structure and intricate functions of our genome. In this Research Topic we wish to explore state-of-the art fluorescence imaging and photomanipulation approaches for the visualization and control of genome organization and function, and the impact on our understanding their disregulation in cancer cells.

At the structural level, super-resolution microscopy enables imaging of individual DNA fibers as well as zooming in on chromatin sub-domains. If combined with in situ hybridization or with live labels for genomic loci these methods can determine how selected DNA sequences are positioned within the three-dimensional chromatin space and how these patterns are affected by signaling cues promoting carcinogenesis. Ultimately, topological signatures associated with disease development may be identified.

The interaction of DNA and chromatin proteins underlying nuclear organization can be directly visualized on isolated, fluorescently-labeled DNA fibers. With the help of microfluidic devices thousands of these fibers can be prepared as arrays enabling studies of DNA-protein interaction in high-throughput. Here, total internal reflection microscopy (TIRF) and stimulated emission depletion (STED) can be used to image proteins at increasing resolution up to single molecules. In combination with optical tweezers to stretch DNA fibers mechanically, these methods can explore how tension affects DNA-protein binding.

Powerful tools for visualizing genome activity often rely on the measurement of fluorescence fluctuations, as performed in fluorescence recovery after photobleaching (FRAP) or its reciprocal technique, fluorescence photoactivation, and fluorescence correlation spectroscopy (FCS). Transcription, as one example, is followed via labeling single RNA molecules with fluorogenic reporters. The amount of transcript, its binding and diffusion behavior as well as the dynamics of polymerases and RNA-binding proteins are determined quantitatively. In the future, these methods may be also be applied to systems in which gene expression is spatially and temporally controlled by light using optogenetic tools, enabling experiments at defined locations within the nucleus and under selected cellular conditions.

A crucial nuclear function widely investigated using fluorescence imaging and photomanipulation approaches is DNA repair. Targeted microirradiation of cell nuclei using e.g. various laser sources is a convenient method to introduce DNA lesions at selected sites. Time-lapse imaging of fluorescently labeled DNA repair factors yields insights into the dynamics of the repair reaction and of the involved repair complexes. Fluorescence fluctuation measurements can be employed to determine kinetic parameters of DNA repair proteins at sites of damage. Moreover, these markers are used to visualize repair foci and study their mobility in the context of different chromatin environments. Finally, super-resolution techniques allow visualization of single DNA strand breaks. By literally shedding light onto the mechanisms responsible for maintaining genome integrity, imaging techniques contribute significantly to advancing our understanding of cancer development.


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