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Strickfaden, Hilmar (2010): Nuclear architecture explored by live-cell fluorescence microscopy using laser and ion microbeam irradiation.. Dissertation, LMU München: Fakultät für Biologie

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Nuclear architecture is a biological field of research that studies the spatio-temporal organization of the components within cell nuclei. Since nuclei are the organelles that harbor the genome and epigenome, they are the place where most of the genetic processes like replication, transcription, splicing, gene-regulation, DNA repair, re- combination etc. are carried out. In the presented doctoral thesis modern 4D live-cell microscopy in combination with laser or ion microbeam irradiation (to label or damage chromatin, respectively) was used to study nuclear architecture in living cells over extended periods of time at the single cell level. The results presented in this thesis can be partitioned into three main parts: (a) chromatin dynamics in cycling cells, (b) adaptation of the ion micro beam facil- ity SNAKE to the needs of live-cell observation (including first experiments) and (c) exploring spatio-temporal dynamics of DNA repair proteins after laser micro ir- radiation. (A) Chromatin dynamics in cycling cells Distribution of interphase chromosomes within cell nuclei has been found to be non- random with respect to gene density and chromosome size. Changes in nuclear orga- nization have been reported in several disorders and diseases. To which extent relative chromosome positioning is conserved through mitosis in cycling cells and whether certain chromatin domains are able change their relative position dramatically in the interphase nucleus has been the subject of various mechanistic models and contro- versial discussions. In 1909 German biologist theodor Boveri was the first one to comment on this topic in his publication: “Die Blastomerenkerne von Ascaris mega- locephala und die Theorie der Chromosomenindividualität” (included as an appendix to this thesis). In order to test Boveri’s hypotheses, 4D live-cell observations were carried out on a modern spinning disc confocal microscope using a human cell line that possesses photoactivatable chromatin. In experiments that used photoactivation and photobleaching of chromatin, it could be demonstrated that – as stated by Boveri – chromatin proximity relationships are in general not conserved through mitosis but destroyed during early prometaphase by the mechanics of mitosis. Other experiments showed that nuclear rotations in a conveyer-belt-like manner are able to bring initially distant chromatin domains into close proximity in a matter of a few minutes. (B) Adaptation of the SNAKE micro beam facility to the needs of live- cell microscopy (including first experiments) Since ordinary irradiation sources lack the ability to perform targeted micro irradia- tion at the micrometer scale and laser micro irradiation produces an artificial mix of various DNA damages, the ion microbeam SNAKE represents an interesting tool to explore the dynamics of repair proteins in a spatio-temporal context. In the course of a collaboration project the ion microbeam was adapted to the needs of long-term live-cell microscopy. These adaptations and first live-cell experiments performed at the refurbished ion micro beam are described in this part of the results. (C) Exploring spatio-temporal dynamics of DNA repair proteins after laser micro irradiation. Mutation of genetic information can cause serious harm to a cell or even a whole or- ganism. DNA repair serves to protect and clean the genome from undirected poten- tially hazardous changes. Compared to the wealth of information which is available about DNA repair at the molecular level only little attention has been payed to it in context of nuclear architecture. In the last part of the results cells stably expressing GFP tagged versions of the repair proteins MDC1, Rad52 and 53BP1 were damaged by laser micro irradiation and imaged over extended periods of time. It could be de- monstrated that at the used damage induction conditions most of the cells show only minor changes with respect to localization of damage signals, kinetochores and nu- cleoli pattern over time. Furthermore, disappearance of spontaneous 53BP1-GFP foci in favor of protein recruitment to damaged chromatin and mutual exclusion between kinetochore signals and Rad52-GFP damage foci could be observed. In a few U2OS Rad52-GFP nuclei DNA damage foci disappeared simultaneously after a dramatic phase in which the total number of foci drastically increased – even adjacent to the laser damaged chromatin.