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Cytomics of nuclear proteins involved in chromatin dynamics, compartmentalisation and genome integrity: integration of single cell analysis in vivo and in silico

Winnok De Vos (UGent)
(2009)
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Promoter
(UGent)
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Abstract
From its first ignorant description as a plain dark spot two centuries ago, the cell nucleus has emerged as the central hub which stores and processes information to coordinate the cell's activities. Ensuring proper functionality, the nucleus is meticulously organized with chromatin as well as proteins occupying distinct but interacting subcompartments. Though organized, the nuclear interior is far from static; all nuclear components demonstrate distinct kinetic behaviour and different levels of mobility, which are crucial for the regulation and coordination of nuclear processes and guarantee flexibility in varying environmental conditions. Disturbance of the spatiotemporal organisation of the nucleus is correlated with aging and disease. This is exemplified by a variety of interrelated nuclear components (Ch. 1). Telomeres, the end structures of mammalian chromosomes, are known to gradually erode with (cellular and organism) age. Proper telomere maintenance is crucial for successful aging as accelerated shortening is observed in premature aging diseases and tumour cells display telomere stabilization or even telomere lengthening. Impaired telomere function leads to nuclear reorganisation, characterized by telomere fusions and consequent genome instability. Defects in architectural components such as the lamina, which delineates the nuclear boundary, elicit a spectrum of diseases, referred to as laminopathies, characterized by dramatic nuclear deformations and reorganisations. Some laminopathies, such as Hutchinson Gilford Progeria Syndrome, lead to premature aging as well and demonstrate accelerated telomere shortening and increased DNA damage. Accumulation of irreparable DNA damage is in turn suspected to contribute to the aging phenotype but also to instigate genome instability. Given the dual role of many of these nuclear processes, it appears that the cell walks a thin line between death and immortality. Understanding this complexity requires sensitive tools that allow for studying nuclear organisation within a natural context. Microscopy has the adequate spatiotemporal resolution to visualize intracellular behaviour. However, current microscopic methods are limited in their possibility of extracting quantitative information and microscopic images are often manually evaluated, which is inevitably prone to variability and subjectivity. The aim of this work was to establish robust methods for objective and quantitative analysis of nuclear organisation and dynamics within the single cell. The act of measuring cellular properties is also referred to as cytometry. In order to provide insights in the nuclear microenvironment we have addressed single- cell analysis from two converging perspectives – in a top-down fashion by means of high content cytometry and bottom-up by 4-dimensional live cell imaging methods. To quantify subcellular characteristics in a population of cells with regard for the stochasticity of events in a natural context, we devised a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear foci using automated fluorescence mosaic microscopy, optimized digital image processing tools and supervised classification feedback (Ch. 2). The HCS approach has a broad application radius and was used inter alia to asses DNA damage in the framework of a space experiment (Ch. 3) as well as to model nuclear shape in cells from patients suffering from laminopathies (Ch. 6). Given the importance of the temporal aspect in nuclear organisation we diverted to live-cell imaging. We focused on telomeres because of their pivotal role in aging and genome stability. To visualize telomeres in living cells we created cell lines stably producing fluorescent telomere-binding fusion proteins. In order to obtain a quantitative signature of the spatiotemporal organisation within the nuclear volume we elaborated an image analysis toolbox that allows for documenting 3- dimensional telomere characteristics and accurately tracking telomeric spots through time (Ch. 4). As we progressed we bumped into another limitation of current imaging systems, which is the limited possibility of observing cells for a long time. This is due to the difficulty of keeping cells alive on the microscope stage and the loss of fluorescent signal or bleaching; both caused by excessive exposure to light. Therefore, novel technology of Controlled Light Exposure Microscopy (CLEM) was applied, which enabled highly resolved long term imaging and reduced photobleaching to a negligable phenomenon. Using this setup, we found that telomeres share small territories at specific positions in the nucleus where they dynamically interact. Most telomeres showed limited motion, but some appeared to move transiently in a directed and energy-dependent manner (Ch. 5). Telomere mobility as well as global nuclear dynamcis were found altered in cells bearing lamin mutations. Absence of functional Lamin A/C, resulted in complete spatiotemporal mayhem in the nucleus and revealed a remarkable transient permeability, indicative of a reduced barrier function of the nuclear envelope. We thereby exposed a novel trait that might underlie the disease mechanism in laminopathies (Ch. 6). In conclusion, studying the cell’s constituents in their natural environment by quantitative means will not only generate fundamental knowledge about their function in the cell, but will also provide insights in the molecular defects underlying disease, which in turn is essential for the development of new diagnostic tools or drugs.
Keywords
cell biology, telomeres, laminopathies, light microscopy, lamina, ageing, image analysis, nucleus

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Chicago
De Vos, Winnok. 2009. “Cytomics of Nuclear Proteins Involved in Chromatin Dynamics, Compartmentalisation and Genome Integrity: Integration of Single Cell Analysis in Vivo and in Silico”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
De Vos, Winnok. (2009). Cytomics of nuclear proteins involved in chromatin dynamics, compartmentalisation and genome integrity: integration of single cell analysis in vivo and in silico. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
De Vos W. Cytomics of nuclear proteins involved in chromatin dynamics, compartmentalisation and genome integrity: integration of single cell analysis in vivo and in silico. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2009.
MLA
De Vos, Winnok. “Cytomics of Nuclear Proteins Involved in Chromatin Dynamics, Compartmentalisation and Genome Integrity: Integration of Single Cell Analysis in Vivo and in Silico.” 2009 : n. pag. Print.
@phdthesis{2096997,
  abstract     = {From its first ignorant description as a plain dark spot two centuries ago, the cell nucleus has emerged as the central hub which stores and processes information to coordinate the cell's activities. Ensuring proper functionality, the nucleus is meticulously organized with chromatin as well as proteins occupying distinct but interacting subcompartments. Though organized, the nuclear interior is far from static; all nuclear components demonstrate distinct kinetic behaviour and different levels of mobility, which are crucial for the regulation and coordination of nuclear processes and guarantee flexibility in varying environmental conditions. Disturbance of the spatiotemporal organisation of the nucleus is correlated with aging and disease. This is exemplified by a variety of interrelated nuclear components (Ch. 1). Telomeres, the end structures of mammalian chromosomes, are known to gradually erode with (cellular and organism) age. Proper telomere maintenance is crucial for successful aging as accelerated shortening is observed in premature aging diseases and tumour cells display telomere stabilization or even telomere lengthening. Impaired telomere function leads to nuclear reorganisation, characterized by telomere fusions and consequent genome instability. Defects in architectural components such as the lamina, which delineates the nuclear boundary, elicit a spectrum of diseases, referred to as laminopathies, characterized by dramatic nuclear deformations and reorganisations. Some laminopathies, such as Hutchinson Gilford Progeria Syndrome, lead to premature aging as well and demonstrate accelerated telomere shortening and increased DNA damage. Accumulation of irreparable DNA damage is in turn suspected to contribute to the aging phenotype but also to instigate genome instability. Given the dual role of many of these nuclear processes, it appears that the cell walks a thin line between death and immortality.
Understanding this complexity requires sensitive tools that allow for studying nuclear organisation within a natural context. Microscopy has the adequate spatiotemporal resolution to visualize intracellular behaviour. However, current microscopic methods are limited in their possibility of extracting quantitative information and microscopic images are often manually evaluated, which is inevitably prone to variability and subjectivity. The aim of this work was to establish robust methods for objective and quantitative analysis of nuclear organisation and dynamics within the single cell. The act of measuring cellular properties is also referred to as cytometry. In order to provide insights in the nuclear microenvironment we have addressed single- cell analysis from two converging perspectives -- in a top-down fashion by means of high content cytometry and bottom-up by 4-dimensional live cell imaging methods.
To quantify subcellular characteristics in a population of cells with regard for the stochasticity of events in a natural context, we devised a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear foci using automated fluorescence mosaic microscopy, optimized digital image processing tools and supervised classification feedback (Ch. 2). The HCS approach has a broad application radius and was used inter alia to asses DNA damage in the framework of a space experiment (Ch. 3) as well as to model nuclear shape in cells from patients suffering from laminopathies (Ch. 6).
Given the importance of the temporal aspect in nuclear organisation we diverted to live-cell imaging. We focused on telomeres because of their pivotal role in aging and genome stability. To visualize telomeres in living cells we created cell lines stably producing fluorescent telomere-binding fusion proteins. In order to obtain a quantitative signature of the spatiotemporal organisation within the nuclear volume we elaborated an image analysis toolbox that allows for documenting 3- dimensional telomere characteristics and accurately tracking telomeric spots through time (Ch. 4). As we progressed we bumped into another limitation of current imaging systems, which is the limited possibility of observing cells for a long time. This is due to the difficulty of keeping cells alive on the microscope stage and the loss of fluorescent signal or bleaching; both caused by excessive exposure to light. Therefore, novel technology of Controlled Light Exposure Microscopy (CLEM) was applied, which enabled highly resolved long term imaging and reduced photobleaching to a negligable phenomenon. Using this setup, we found that telomeres share small territories at specific positions in the nucleus where they dynamically interact. Most telomeres showed limited motion, but some appeared to move transiently in a directed and energy-dependent manner (Ch. 5). Telomere mobility as well as global nuclear dynamcis were found altered in cells bearing lamin mutations. Absence of functional Lamin A/C, resulted in complete spatiotemporal mayhem in the nucleus and revealed a remarkable transient permeability, indicative of a reduced barrier function of the nuclear envelope. We thereby exposed a novel trait that might underlie the disease mechanism in laminopathies (Ch. 6).
In conclusion, studying the cell{\textquoteright}s constituents in their natural environment by quantitative means will not only generate fundamental knowledge about their function in the cell, but will also provide insights in the molecular defects underlying disease, which in turn is essential for the development of new diagnostic tools or drugs.},
  author       = {De Vos, Winnok},
  isbn         = {9789059893030},
  keyword      = {cell biology,telomeres,laminopathies,light microscopy,lamina,ageing,image analysis,nucleus},
  language     = {eng},
  pages        = {V, 199},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {Cytomics of nuclear proteins involved in chromatin dynamics, compartmentalisation and genome integrity: integration of single cell analysis in vivo and in silico},
  year         = {2009},
}