Microtubules are essential for many dynamic processes occurring within eukaryotic cells including organelle and vesicular trafficking, motility of cilia and flagella, and mitosis. Microtubules operate in conjunction with the kinesin superfamily of microtubule-dependent motor proteins, which use the energy from ATP hydrolysis to "walk" along microtubule tracks, and in doing so generate force for the transport of cellular cargo and mitosis. The goal of this project was to define the microtubule interactions and regulation of two kinesin-like proteins (KLPs), the Homo sapiens mitotic kinesin-like protein-1 (HsMKLP-1) and the Arabidopsis thaliana kinesin-like calmodulin-binding protein (KCBP). Functional domains of HsMKLP-1 and KCBP were heterogeneously expressed in insect cells (HsMKLP-1) and/or E. coli (HsMKLP-1, KCBP) and used to examine the microtubule binding and ATPase activity of HsMKLP-1 and KCBP catalytic domains. Overall, the HsMKLP-1 catalytic domain was found to operate in a similar fashion to other KLPs with respect to microtubule binding and ATP hydrolysis, but HsMKLP-1 exhibited enhanced microtubule binding of the dimer and weaker affinity for ATP that functionally distinguishes it from other KLPs. HsMKLP-1 proteins were also used to generate HsMKLP-1 specific antibodies to be used as a tool for characterizing native HsMKLP-1. To define the role of nuclear localization in regulating the activity of HsMKLP-1 during interphase, sequences directing nuclear localization of HsMKLP-1 were identified. Mutation of the nuclear localization sequence 799PNGSRKRR806 to 799PNGSRTSR806 or removal of AA's 830-856 of HsMKLP-1, which contains the nuclear localization sequence 851PKRKKP856, were sufficient to abolish nuclear localization. In the absence of a functional nuclear localization sequence HsMKLP-1 localized to microtubule plus ends, suggesting that nuclear localization serves to limit the interaction of HsMKLP-1 with the interphase microtubule array. The KCBP catalytic domain, which contains a calmodulin-binding site, was used to determine the effect of Ca2+/calmodulin on the microtubule binding and ATPase activity of KCBP. Ca2+/calmodulin was found to inhibit the binding of KCBP to microtubules and reduced the motor's microtubule-stimulated ATPase activity, which suggests that Ca2+/calmodulin may modulate the activity of KCBP in vivo by regulating the motor's association with microtubules. / Ph. D.

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.