Structure and Dynamics of Replication Domains in Single Chromosome Territories of Interphase Nuclei

Knowing the three-dimensional organization of chromatin sets the framework for understanding genome regulation. Our picture of higher order chromatin structure insitu however remains fragmentary at many scales, since it is not directly accessible by imaging technologies available today. The recently revealed domain organization of chromatin subunits into sub-megabasepair sized topologically associating domains (TADs), enabled by chromosome conformation capture based techniques, marks a significant advancement in understanding chromatin architecture. Similarly quantitative methods for the analysis of global structure and dynamics of chromatin in single living cells are currently lacking, leaving it unclear how TADs are manifested within a single nucleus and how dynamic topological chromatin interactions are in living cells. To start to address this gap in our knowledge, I set out to systematically probe the basic polymer features of chromatin at the level of replication domains (RDs) in single cells as a basis for a model of higher order chromatin organization. I have addressed both structural and dynamic aspects of RD organization during interphase. Using super-resolution microscopy, I was able to investigate RD organization at unprecedented resolution. I found that the median RD diameter is ~150 nm, significantly smaller than the ~270 nm distance to the nearest neighbor, which leaves sufficient physical space for extended linker regions between RDs. By quantifying correlated motion of neighboring RDs, I could reveal the typical elastic coupling range between RDs to be ~500 nm. Combining super-resolution microscopy with a perturbation experiment I could further obtain evidence for the model that chromatin compaction upon ATP depletion is predominantly mediated by preferential compaction of linker regions between RDs, rather than by compaction of RDs themselves. In addition to these structural parameters of RD organization, I also characterized the diffusional behavior of interphase RDs of single chromosome territories. Tracking 1,372 RDs of 141 chromosome territories allowed me to obtain a global and statistically robust view of interphase chromatin dynamics across the entire nucleus. My data confirms that heterochromatin chromatin is immobile within a few hundred nanometers of the nuclear membrane and nucleolar surface over the time scale of several minutes and that nucleoplasmic dynamics is characterized by anomalous diffusion. I did not observe reproducible directed motion of RDs on the timescale of seconds to a minute. I observed a systematic reduction in chromatin motion as the cell cycle progressed from G1 to late S-phase and an increase in mobility if I artificially increased nuclear volume by allowing cells to grow when DNA replication was inhibited. My observations on native and perturbed chromatin structure and dynamics in nuclei of living cells allow me to propose a comprehensive model of higher order chromatin organization in single cells, that consists of stable structuring units of RDs, which are connected by extended flexible linker domains, whose dynamics are limited by attachment to the nuclear periphery and nucleoli and the available free volume inside the nucleus

Biological Protein Patterning Systems across the Domains of Life: from Experiments to Modelling

Distinct localisation of macromolecular structures relative to cell shape is a common feature across the domains of life. One mechanism for achieving spatiotemporal intracellular organisation is the Turing reaction-diffusion system (e.g. Min system in the bacterium Escherichia coli controlling in cell division). In this thesis, I explore potential Turing systems in archaea and eukaryotes as well as the effects of subdiffusion. Recently, a MinD homologue, MinD4, in the archaeon Haloferax volcanii was found to form a dynamic spatiotemporal pattern that is distinct from E. coli in its localisation and function. I investigate all four archaeal Min paralogue systems in H. volcanii by identifying four putative MinD activator proteins based on their genomic location and show that they alter motility but do not control MinD4 patterning. Additionally, one of these proteins shows remarkably fast dynamic motion with speeds comparable to eukaryotic molecular motors, while its function appears to be to control motility via interaction with the archaellum. In metazoa, neurons are highly specialised cells whose functions rely on the proper segregation of proteins to the axonal and somatodendritic compartments. These compartments are bounded by a structure called the axon initial segment (AIS) which is precisely positioned in the proximal axonal region during early neuronal development. How neurons control these self-organised localisations is poorly understood. Using a top-down analysis of developing neurons in vitro, I show that the AIS lies at the nodal plane of the first non-homogeneous spatial harmonic of the neuron shape while a key axonal protein, Tau, is distributed with a concentration that matches the same harmonic. These results are consistent with an underlying Turing patterning system which remains to be identified. The complex intracellular environment often gives rise to the subdiffusive dynamics of molecules that may affect patterning. To simulate the subdiffusive transport of biopolymers, I develop a stochastic simulation algorithm based on the continuous time random walk framework, which is then applied to a model of a dimeric molecular motor. This provides insight into the effects of subdiffusion on motor dynamics, where subdiffusion reduces motor speed while increasing the stall force. Overall, this thesis makes progress towards understanding intracellular patterning systems in different organisms, across the domains of life

STED Nanoscopy to Illuminate New Avenues in Cancer Research – From Live Cell Staining and Direct Imaging to Decisive Preclinical Insights for Diagnosis and Therapy

Molecular imaging is established as an indispensable tool in various areas of cancer research, ranging from basic cancer biology and preclinical research to clinical trials and medical practice. In particular, the field of fluorescence imaging has experienced exceptional progress during the last three decades with the development of various in vivo technologies. Within this field, fluorescence microscopy is primarily of experimental use since it is especially qualified for addressing the fundamental questions of molecular oncology. As stimulated emission depletion (STED) nanoscopy combines the highest spatial and temporal resolutions with live specimen compatibility, it is best-suited for real-time investigations of the differences in the molecular machineries of malignant and normal cells to eventually translate the acquired knowledge into increased diagnostic and therapeutic efficacy. This thesis presents the application of STED nanoscopy to two acute topics in cancer research of direct or indirect clinical interest. The first project has investigated the structure of telomeres, the ends of the linear eukaryotic chromosomes, in intact human cells at the nanoscale. To protect genome integrity, a telomere can mask the chromosome end by folding back and sequestering its single-stranded 3’-overhang in an upstream part of the double-stranded DNA repeat region. The formed t-loop structure has so far only been visualized by electron microscopy and fluorescence nanoscopy with cross-linked mammalian telomeric DNA after disruption of cell nuclei and spreading. For the first time, this work demonstrates the existence of t-loops within their endogenous nuclear environment in intact human cells. The identification of further telomere conformations has laid the groundwork for distinguishing cancerous cells that use different telomere maintenance mechanisms based on their individual telomere populations by a combined STED nanoscopy and deep learning approach. The population difference was essentially attributed to the promyelocytic leukemia (PML) protein that significantly perturbs the organization of a subpopulation of telomeres towards an open conformation in cancer cells that employ a telomerase-independent, alternative telomere lengthening mechanism. Elucidating the nanoscale topology of telomeres and associated proteins within the nucleus has provided new insight into telomere structure-function relationships relevant for understanding the deregulation of telomere maintenance in cancer cells. After understanding the molecular foundations, this newly gained knowledge can be exploited to develop novel or refined diagnostic and treatment strategies. The second project has characterized the intracellular distribution of recently developed prostate cancer tracers. These novel prostate-specific membrane antigen (PSMA) inhibitors have revolutionized the treatment regimen of prostate cancer by enabling targeted imaging and therapy approaches. However, the exact internalization mechanism and the subcellular fate of these tracers have remained elusive. By combining STED nanoscopy with a newly developed non-standard live cell staining protocol, this work confirmed cell surface clustering of the targeted membrane antigen upon PSMA inhibitor binding, subsequent clathrin-dependent endocytosis and endosomal trafficking of the antigen-inhibitor complex. PSMA inhibitors accumulate in prostate cancer cells at clinically relevant time points, but strikingly and in contrast to the targeted antigen itself, they eventually distribute homogenously in the cytosol. This project has revealed the subcellular fate of PSMA/PSMA inhibitor complexes for the first time and provides crucial knowledge for the future application of these tracers including the development of new strategies in the field of prostate cancer diagnostics and therapeutics. Relying on the photostability and biocompatibility of the applied fluorophores, the performance of live cell STED nanoscopy in the field of cancer research is boosted by the development of improved fluorophores. The third project in this thesis introduces a biocompatible, small molecule near-infrared dye suitable for live cell STED imaging. By the application of a halogen dance rearrangement, a dihalogenated fluorinatable pyridinyl rhodamine could be synthesized at high yield. The option of subsequent radiolabeling combined with excellent optical properties and a non-toxic profile renders this dye an appropriate candidate for medical and bioimaging applications. Providing an intrinsic and highly specific mitochondrial targeting ability, the radiolabeled analogue is suggested as a vehicle for multimodal (positron emission tomography and optical imaging) medical imaging of mitochondria for cancer diagnosis and therapeutic approaches in patients and biopsy tissue. The absence of cytotoxicity is not only a crucial prerequisite for clinically used fluorophores. To guarantee the generation of meaningful data mirroring biological reality, the absence of cytotoxicity is likewise a decisive property of dyes applied in live cell STED nanoscopy. The fourth project in this thesis proposes a universal approach for cytotoxicity testing based on characterizing the influence of the compound of interest on the proliferation behavior of human cell lines using digital holographic cytometry. By applying this approach to recently developed live cell STED compatible dyes, pronounced cytotoxic effects could be excluded. Looking more closely, some of the tested dyes slightly altered cell proliferation, so this project provides guidance on the right choice of dye for the least invasive live cell STED experiments. Ultimately, live cell STED data should be exploited to extract as much biological information as possible. However, some information might be partially hidden by image degradation due the dynamics of living samples and the deliberate choice of rather conservative imaging parameters in order to preserve sample viability. The fifth project in this thesis presents a novel image restoration method in a Bayesian framework that simultaneously performs deconvolution, denoising as well as super-resolution, to restore images suffering from noise with mixed Poisson-Gaussian statistics. Established deconvolution or denoising methods that consider only one type of noise generally do not perform well on images degraded significantly by mixed noise. The newly introduced method was validated with live cell STED telomere data proving that the method can compete with state-of-the-art approaches. Taken together, this thesis demonstrates the value of an integrated approach for STED nanoscopy imaging studies. A coordinated workflow including sample preparation, image acquisition and data analysis provided a reliable platform for deriving meaningful conclusions for current questions in the field of cancer research. Moreover, this thesis emphasizes the strength of iteratively adapting the individual components in the operational chain and it particularly points towards those components that, if further improved, optimize the significance of the final results rendering live cell STED nanoscopy even more powerful

Segmentation of heterochromatin foci using a 3D spherical harmonics intensity model.

Abstract. We introduce a 3D model-based approach for automatic segmentation of 3D fluorescent heterochromatin foci from 3D microscopy images. The approach employs a new 3D parametric intensity model based on a spherical harmonics (SH) expansion and can represent foci of regular and highly irregular shapes. By solving a least-squares optimization problem, the model is directly fitted to the 3D image data, and the model parameters including the SH expansion coefficients are estimated. The approach has been successfully applied to real 3D microscopy image data. A visual comparison and a quantitative evaluation show that the new approach yields better results than previous approaches

Mechanical investigation of wall-less plant cells using microfluidics

Each cell within a plant experiences a myriad of mechanical and chemical cues that direct growth and development. Selective application of such cues and live-imaging of the resulting cellular responses are challenging within the tissue context. For this reason, I have developed a microfluidic device, called Ψ-trap, for mechano-chemical investigations of single cells, by combining microfluidic technology and automated live-imaging. The platform allows the microscopic time-course observation of individual plant cells within a heterogeneous population, such as leaf cells, while applying precise chemical or physical stimuli. As a pilot study I have quantitatively monitored the cellular expansion of cell wall-less plant cells, called protoplasts. I have further shown that the application of cyclic compression forces to single cells inside the Ψ-trap can be used to study the mechanical volume and shape control of single protoplasts from different developmental conditions. To separate the intracellular and extracellular factors that influence the cellular shape control, I have created a complementary microfluidic shape induction device, called the Ψ-constriction trap. My results suggest wall-less shape retainment upon mechanical compression in plant cells

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin