Proper cell division is a fundamental process for the development and sustainability of healthy living organisms. Defective cell division can have deleterious effects on tissue homeostasis and can represent the first step towards disease development. The overall goal of this work was to develop and validate a new, mitosis-based, single-cell toxicity approach. This contributes to the current need of toxicology research to replace animal testing with predictive in vitro models. Cell division-based assays would be better at predicting risk than other commonly used in vitro measurements, such as persistent cell cycle arrest or cell death. Finally, single-cell microscopic analysis provides far deeper insight into the underlying toxicity mechanism(s) than bulk cell population measurements. To meet our goal, we investigated the toxicity of silver nanoparticles (AgNPs) on immortalized human retinal pigmented epithelial (RPE-1) cells. AgNPs are a major nanomaterial employed in product manufacturing due to desirable antimicrobial properties, yet toxicity reports are still confounding. RPE-1 cells were cultured in the presence of low and high doses of polyvinylpyrrolidone (PVP)-coated AgNPs for a single 24-hour treatment (acute treatment), for six 24-hour treatments administered over a period of 3 weeks (moderate treatment), or for twelve 24-hour treatments administered over a period of 6 weeks (chronic treatment). Time-lapse, phase-contrast microscopy of acutely treated cells showed that 100% of cells engulfed AgNPs, which was further confirmed by electron microscopy. Moreover, we found that higher concentrations of AgNPs resulted in large numbers of acutely treated cells becoming arrested in mitosis, dying, or dividing abnormally. In contrast, untreated cells displayed normal mitotic behavior. High-resolution fluorescence microscopy performed in treated cell populations identified an increased percentage of abnormal nuclear morphologies compared to the untreated cells. Further live-cell analysis indicated that treated cells failed cytokinesis or slipped out of mitosis more often than untreated cells. Overall, our results indicate that AgNPs impair cell division, not only further confirming toxicity to human cells, but also revealing previously unreported toxicity mechanisms and highlighting the propagation of adverse phenotypes within the cell population after exposure. Furthermore, this work illustrates that cell division-based single-cell analysis could provide an alternative to animal experimentation in the future. / Master of Science / Multiple agencies, including the U.S. Environmental Protection Agency and the National Academy of Science, are urging for a radical paradigm shift from standard, whole-animal testing to alternative and novel technologies. To meet this urgent need, we aimed to develop a new, cell division-focused toxicity assay by investigating the mechanism of toxicity from silver nanoparticles (AgNPs) on human retinal pigment epithelial (RPE-1) cells. Cultured RPE-1 cells were treated with varying concentrations of AgNPs and live-cell microscopy was used to analyze the behavior of cells undergoing cell division over a 24 hour time period. Physical interaction between cells and particles was visually observed and 100% of treated cells appeared to engulf particles. We found that higher concentrations of AgNPs resulted in large numbers of cells stalling in mitosis and/or dying. In contrast, untreated cells displayed normal mitotic behavior. High-resolution fluorescence microscopy performed in chronically treated cell populations identified an increased percentage of binucleated cells. Further live-cell analysis indicated that one major cell division defect could explain the binucleated cell phenotype. Indeed, treated cells failed cytokinesis (cytoplasmic division following mitotic chromosome segregation) more often than control cells. Overall, our results indicate that AgNPs specifically impair cell division, not only further confirming toxicity to human cells, but also revealing specific, previously unreported toxicity mechanisms and highlighting the propagation of adverse phenotypes within the cell population after exposure. Furthermore, this work illustrates that cell division-based assays and ingle-cell analysis could greatly benefit chemical safety experimentation in the future.

Despite the importance of zinc in cell proliferation and development, mechanisms of zinc redistribution during these processes remain largely elusive. Given the limited external supply of nutrients during embryogenesis, developing organs most likely redistribute zinc from neighboring cells to satisfy their increased demand, thus raising the intriguing and fundamental question of how the limited supply of zinc in a fertilized egg is redistributed in the course of embryonic development. To systematically explore this question, we employed both cell culture and zebrafish as model systems in combination with a Zn(II)-selective fluorescent probe and synchrotron X-ray fluorescence (SXRF) microtomography studies. Using the Zn(II)-selective emission ratiometric fluorescent probe designed in our lab, we followed the redistribution dynamics of labile Zn(II) pools in a zebrafish embryo during the first 24 hours post fertilization. Furthermore, SXRF microtomography studies were used to visualize the 3D distribution of total zinc in fixed zebrafish samples. From this method we successfully reconstructed a 3D elemental distribution map at 5 μm resolution. The volumetric map revealed a distinct zinc distribution that could be correlated with characteristic anatomical features at this stage of embryonic development. Together these powerful techniques allow us to study both labile zinc in live samples and total zinc content in fixed samples in order to achieve a more detailed understanding of the zinc redistribution dynamics during embryogenesis.

A large brain is one of the most distinguishing features of humans compared to other members of the animal kingdom. During mammalian evolution there has been a disproportionate enlargement of the brain relative to body size and this expansion has been particularly prominent during the past 3 million years of human lineage. This must be the consequence of adaptive genetic alterations during mammalian evolution, but the genes and molecular processes altered are essentially unknown. One approach for identifying candidate genes for brain size regulation is through characterisation of Mendelian disorders of brain development. In particular, primary microcephaly has received considerable interest as a model disease for studying brain size regulators because patients present with a profoundly reduced brain size but have no other malformations. Genetic studies have identified mutations in seven genes that can cause primary microcephaly. All the primary microcephaly proteins localise to the centrosome at some stage during the cell cycle and have roles in a diverse range of functions including centrosome maturation, centriole formation and microtubule organisation at the spindle pole. The precise mechanism leading to primary microcephaly is not known but a prevalent hypothesis is that centrosome dysfunction disrupts mitosis of neural progenitor cells. Despite there being strong evidence in support of this hypothesis for most primary microcephaly genes, MCPH1 (the first primary microcephaly gene to be identified) always appeared to be functionally distinct from other primary microcephaly proteins. Most work on MCPH1 has focussed on its role in the DNA damage response and cell cycle timing rather than on its mitotic role. As a result, the aim of this thesis is to perform a detailed analysis of MCPH1 function during mitosis. In this thesis, three isoforms of MCPH1 were characterised and their localisation, expression and stability examined. It was established that MCPH1 is highly regulated during mitosis. MCPH1 transcript and protein levels vary significantly throughout the cell cycle and MCPH1 protein is targeted for degradation late in mitosis. In addition, MCPH1 is hyperphosphorylated during mitosis (in prometaphase-arrested cells) suggesting that phosphorylation could potentially regulate MCPH1 mitotic function. Twelve mitotic phosphorylation sites were identified by phosphopeptide mapping, many of which were CDK1 and PLK1 consensus sites. Both PLK1 and CDK1 also contribute to MCPH1 phosphorylation in vivo. Although MCPH1 non-phosphorylatable mutants localise normally during mitosis, binding to interaction partners may be affected which may have functional consequences. During mitosis MCPH1 localises to the centrosomes and kinetochores. Consistent with this localisation, RNAi-mediated knockdown of MCPH1 leads to metaphase arrest with multipolar spindles, major defects in chromosome alignment and loss of chromatid cohesion. In addition, MCPH1 deficient mouse embryonic fibroblast cells also demonstrate similar chromosome alignment defects, strengthening this finding in an independent system. Live-imaging of MCPH1 depleted cells demonstrate that a normal bipolar spindle and metaphase plate are initially formed, but subsequently chromosomes and chromatids drop off the metaphase plate and eventually the spindle collapses. This suggests that the primary function of MCPH1 is to allow timely progression through metaphase, possibly by mediating kinetochore-microtubule attachments to satisfy the spindle activated checkpoint. Therefore my work describes several roles for MCPH1 in mitosis (centrosome stability, chromosome alignment and metaphase progression) suggesting that its role in mitosis could result in primary microcephaly in a number of different ways.

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Thesis advisor: Charles Hoffman / Faithful chromosome segregation is necessary for the successful completion of mitosis and meiosis. The centromere is the site of kinetochore and microtubule attachment during chromosome segregation, and it is critical that the centromere is properly formed and maintained. Many proteins contribute to centromere formation, and this process has been extensively studied during the mitotic cell cycle. However, the roles of the centromere and its associated proteins during meiosis and their contribution to the fidelity of chromosome segregation process are not as well understood. Here, I aim to elucidate a mechanism that may contribute to aneuploidy in gametes, which is a major contributing factor in human infertility. In this study, I investigate the role of Abp1, the most prominent member of the transposase-derived protein family homologous to mammalian CENP-B in the assembly of centromeric chromatin during meiosis in the fission yeast Schizosaccharomyces pombe. I reveal that in contrast to its known role as a major regulator of LTR retrotransposons during the mitotic and meiotic cell cycles, Abp1 has a specialized role at the centromere during meiosis. My results indicate that Abp1 displays dynamic localization to the centromeres during meiosis compared to the vegetative cell cycle. I show that loss of abp1 impairs pericentromeric heterochromatin and the localization of Cnp1, a CENP-A ortholog, to the centromere central cores during meiosis. Moreover, Abp1 appears to suppress formation of meiotic neocentromeres by restricting deposition of Cnp1 at certain heterochromatin loci. Loss of abp1 has a drastic effect on chromosome segregation, resulting in dramatic frequency of aneuploidy. Furthermore, the genome surveillance role for retrotransposons by Abp1 appears to encompass centromeres as the mere insertion of an LTR sequence within the centromere central cores further exacerbates incidence of meiotic aneuploidy in abp1 null cells. This study provides intriguing insights into factors controlling the assembly of centromeric chromatin and its impact on the fidelity of chromosome segregation process during meiosis with important implications for advancing our understanding of the evolutionary forces driving the evolution of eukaryotic centromeres. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Equal partitioning of genetic materials of the chromosomes is key to the mitotic cell cycle, as unequal segregation of chromosomes during mitosis leads to aneuploidy, a hall mark of human cancer. Accurate chromosome segregation is directed by the kinetochore, a proteinaceous structure on each sister chromosome that physically connects the chromosome to the spindle microtubules. Kinetochore assembles at the centromere, a specialized chromosome region epigenetically defined by the histone H3 variant centromere protein A (CENP-A) in higher eukaryotes including mammals. In order to maintain centromere identity against CENP-A dilution caused by S phase genome replication, new CENP-A molecules are loaded at preexisting centromeres in G1 phase of the cell cycle. Despite of the several important stages and molecular components identified in CENP-A replenishment, little is known about how new CENP-A proteins become stably incorporated into centromeric nucleosomes. Here by using quantitative imaging, pulse-chase labeling, mutant analysis, cellular fractionation and computational simulations, I have identified the cytoskeleton protein diaphanous formin mDia2 to be essential for the essential for the stable incorporation of newly synthesized CENP-A at the centromere. The novel function of mDia2 depends on its nuclear localization and its actin nucleation activity. Furthermore, mDia2 functions downstream of a small GTPase molecular switch during CENP-A loading, and is responsible for the formation of dynamic and short actin filaments observed in early G1 nuclei. Importantly, the maintenance of centromeric CENP-A levels requires a pool of polymerizable actin inside the nucleus. Single particle tracking and quantitative analysis revealed that centromere movement in early G1 nuclei is relatively confined over the time scale of initial CENP-A loading, and the subdiffusive behavior was significantly altered upon mDia2 knockdown. Finally, knocking down mDia2 results in prolonged centromere association of Holliday junction recognition protein (HJURP), a chaperone required to undergo timely turnover to allow for new CENP-A loading at the centromere. Our findings suggest that diaphanous formin mDia2 forms a link between the upstream small GTPase signaling and the downstream confined viscoelastic nuclear environment, and therefore regulates the stable assembly of new CENP-A containing nucleosomes to mark centromeres’ epigenetic identity (Chapter 2 and 3). While centromere identity is essential for kinetochore assembly, once kinetochores are assembled, fine-tuned interactions between kinetochores and microtubules become important for a fully functioning mitotic spindle during chromosome segregation. It has been previously found that another diaphanous formin protein mDia3 and its interaction with EB1, a microtubule plus-end tracking protein, are essential for accurate chromosome segregation1. In Chapter 4 of this thesis, I found that knocking down mDia3 caused a compositional change at the microtubule plus-end attached to the kinetochores, marked by a loss of EB1 and a gain of CLIP-170 and the dynein light chain protein Tctex-1. Interestingly, this compositional change does not affect the release of cytoplasmic dynein from aligned kinetochores, suggesting a population of Tctex-1 can be recruited to the kinetochores without dynein. During mitosis, Tctex-1 associates with unattached kinetochores and is required for accurate chromosome segregation. Tctex-1 knockdown in cells does not affect the localization and function of dynein at the kinetochore, but produces a prolonged mitotic arrest with a few misaligned chromosomes, which are subsequently missegregated during anaphase. This function is independent of Tctex-1’s association with dynein. The kinetochore localization of Tctex-1 is independent of the ZW10-dynein pathway, but requires the Ndc80 complex. Thus, our findings reveal a dynein independent role of Tctex-1 at the kinetochore to enhance the stability of kinetochore-microtubule attachment. Together, these work suggest novel regulatory roles of the cytoskeletal systems in the maintenance as well as subsequent functions of the centromere/kinetochore, and provide mechanistic insights into the complex control principles of accurate chromosome segregation. Our findings provide a new model in understanding the epigenetic maintenance of genome integrity, and will have implications with regard to how aberrant cell divisions underlying aneuploidy can be targeted in the treatment of cancer.

Includes bibliographical references (p. 118-134).

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Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 111-120). Also available on the Internet.