Characterization of chromatin mobility upon DNA damage in Arabidopsis thaliana

Plant cells are subject to high levels of DNA damage from dependence on sunlight for energy and the associated exposure to biotic and abiotic stresses. Double-strand breaks (DSBs) are a particularly deleterious type of DNA damage, potentially leading to chromosome rearrangements or loss of entire chromosome arms. The presence of efficient and accurate repair mechanisms may be particularly important for sedentary organisms with late separation of the germline, such as plants. DSB repair is accomplished by two main pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is achieved by stabilization and re-ligation of broken DNA ends, often with a loss or mutation of bases. HR is a more complex and conservative mechanism in which intact homologous regions are used as a template for repair. The molecular mechanisms that control DSB signaling and repair have been characterized extensively. Nonetheless, little is known about how the homology search happens in the crowded space of the cell nucleus. This thesis reveals the methodology to capture chromatin motion to investigate nuclear dynamics in different developmental and cellular contexts. Using live imaging approaches, we measured chromosome mobility by tracking the motion of specific loci using the lacO/LacI and ParB/parS tagging systems in Arabidopsis thaliana.Our results have shown that chromatin mobility is affected by cell differentiation level, cell cycle phase, or genomic position, and that chromatin mobility increases when DNA damage is induced. Moreover, we observed an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. Importantly, this increase in mobility in S/G2 was lost on sog1-1 mutant, a central transcription factor of the DNA damage response (DDR), indicating that repair mechanisms actively regulate chromatin mobility upon DNA damage. Studies have shown that HR is the predominant DSB repair pathway occurring during S/G2 phase. Therefore, we investigated the mobility of two GFP-tagged HR regulators, RAD51 and RAD54, corresponding to early and late HR. DSB sites show remarkably high mobility levels at the early HR stage. Subsequently, a drastic decrease in DSB mobility is observed, which seems to be associated with the relocation of DSBs to the nucleus periphery.Altogether, our study suggests chromatin mobility as a non-negligible factor for DNA repair in plants, which may facilitate physical searching in the nuclear space thereby helping to locate a homologous template during homology-directed DNA repair

Live-cell visualisation of chromatin for the study of radiation-induced chromosome aberrations

This thesis was submitted for the degree of Master of Philosophy and awarded by Brunel University.Chromosomes can be visualised during the metaphase stage of the cell cycle however, there are various limitations associated with the visualisation of chromatin during interphase. This is due to the fact that during that stage of the cell cycle, chromatin is decondensed and difficult to resolve by using standard techniques. The employment of fluorescence microscopy has provided great insight into the examination of the structure and dynamics of fluorescently tagged chromatin regions in living cells during interphase. Thus the in vivo stable expression of fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) has the potential to address questions that lead to understanding dynamic responses of chromatin in response to DNA damage. In this study, chromatin sites within the nuclei of Chinese Hamster Ovary (CHO) cells were fluorescently labelled using the lac operon system, which works on the principle that EGFP-lac repressor protein will bind to sites of integrated lac operator sequence. To achieve this, CHO cells were transfected with pSV2-DHFR8.32 (lac operator) and p3‟SSdimerClonEGFP (lac repressor-EGFP fusion protein) plasmids by calcium phosphate transfection. 68 stable transfectants were stored and clone CHO-R-O-25 cells were used for further experimental analysis based on these cells containing discrete and intense fluorescent spots. It was shown that the physiological and functional characteristics of CHO-R-O-25 cells were comparable to those of control CHO DG44 cells. Time-lapse fluorescence microscopy was used to examine for any alterations in the physical structure of chromatin in response to DNA damage that was induced by radiation and a range of different chemicals. It was demonstrated that in cells exposed to DNA damaging agents, chromatin diffusion and expansions revealed by alterations in the intensity and size of the fluorescent spots within the nuclei of cells are owed to relaxation of chromatin structures in response to genome insults. The potential for this tool to study the organisational responses of chromatin in response to damage and the ulitimate relevance of any physical alterations to mechanisms for chromosome exchange formation, are described

TALE-mediated plant genome visualization

Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in subnuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms

Insights into nuclear organization in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors

Serine/arginine-rich (SR) proteins are splicing regulators that share a modular structure consisting of one or two N-terminal RNA recognition motif domains and a C-terminal RS-rich domain. We investigated the dynamic localization of the Arabidopsis thaliana SR protein RSZp22, which, as we showed previously, distributes in predominant speckle-like structures and in the nucleolus. To determine the role of RSZp22 diverse domains in its nucleolar distribution, we investigated the subnuclear localization of domain-deleted mutant proteins. Our results suggest that the nucleolar localization of RSZp22 does not depend on a single targeting signal but likely involves different domains/motifs. Photobleaching experiments demonstrated the unrestricted dynamics of RSZp22 between nuclear compartments. Selective inhibitor experiments of ongoing cellular phosphorylation influenced the rates of exchange of RSZp22 between the different nuclear territories, indicating that SR protein mobility is dependent on the phosphorylation state of the cell. Furthermore, based on a leptomycin B- and fluorescence loss in photobleaching-based sensitive assay, we suggest that RSZp22 is a nucleocytoplasmic shuttling protein. Finally, with electron microscopy, we confirmed that RSp31, a plant-specific SR protein, is dynamically distributed in nucleolar cap-like structures upon phosphorylation inhibition. Our findings emphasize the high mobility of Arabidopsis SR splicing factors and provide insights into the dynamic relationships between the different nuclear compartments

Systems microscopy approaches to understand cancer cell migration and metastasis

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration

Investigating the 3D chromatin architecture with fluorescence microscopy

Chromatin is an assembly of DNA and nuclear proteins, which on the one hand has the function to properly store the 2 meters of DNA of a diploid human nucleus in a small volume and on the other hand regulates the accessibility of specific DNA segments for proteins. Many cellular processes like gene expression and DNA repair are affected by the three-dimensional architecture of chromatin. Cohesin is an important and well-studied protein that affects three-dimensional chromatin organization. One of the functions of this motor protein is the active generation of specific domain structures (topologically associating domains (TADs)) by the process of loop extrusion. Studies of cohesin depleted cells showed that TAD structures were lost on a population average. Due to this finding, the question arose, to what extent the functional nuclear architecture, that can be detected by confocal and structured illumination microscopy, is impaired when cells were cohesin depleted. The work presented in this thesis could show that the structuring of the nucleus in areas with different chromatin densities including the localization of important nuclear proteins as well as replication patterns was retained. Interestingly, cohesin depleted cells proceeded through an endomitosis leading to the formation of multilobulated nuclei. Obviously, important structural features of chromatin can form even in the absence of cohesin. In the here presented work, fluorescence microscopic methods were used throughout, and an innovative technique was developed, that allows flexible labeling of proteins with different fluorophores in fixed cells. With this technique DNA as well as peptide nucleic acid (PNA) oligonucleotides can be site-specifically coupled to antibodies via the Tub-tag technology and visualized by complementary fluorescently labeled oligonucleotides. The advantages and disadvantages of PNAs as docking strands are discussed in this thesis as well as the use of PNAs in fluorescence in situ hybridization (FISH). In the next study, which is part of this work, a combination of FISH and super-resolution microscopy was used. There it could be shown that DNA segments of 5 kb can form both compact and elongated configurations in regulatory active as well as inactive chromatin. Coarse-grained modeling of these microscopic data, in agreement with published data from other groups, has suggested that elongated configurations occur more frequently in DNA segments in which the occupancy of nucleosomes is reduced. The microscopically measured distance distributions could only be simulated with models that assume different densities of nucleosomes in the population. Another result of this study was that inactive chromatin - as expected - shows a high level of compaction, which can hardly be explained with common coarse-grained models. It is possible that environmental effects that are difficult to simulate play a role here. Chromatin is a highly dynamic structure, and its architecture is constantly changing, be it through active processes such as the effect of cohesin investigated here or through thermodynamic interactions of nucleosomes as they are simulated in coarse-grained models. It will take a long time until we adequately understand these dynamic processes and their interplay