The development and maintenance of multicellular organisms depends fundamentally on cell division, a series of events largely mediated by the mitotic spindle. Errors in spindle formation and/or function are often associated with severe consequences, most notably cancer. In order to elucidate the cause of such errors and the potential for therapeutic intervention, it is imperative to attain a clear understanding of how cell division normally operates. In this regard, this dissertation focuses on the activity of two microtubule-based motor proteins, Eg5 and dynein, prior to and immediately following nuclear envelope breakdown during mitosis. I show that prophase microtubules are remarkably more dynamic than their metaphase counterparts, moving both toward and away from centrosomes across a wide distribution of rates. Inhibition of Eg5, dynein and Kif2a revealed that a subset of this motion is consistent with microtubule flux, a well-established phenomenon temporally limited to metaphase and anaphase spindles by the preceding literature. My data indicates that flux is operational throughout all of mitosis, possibly functioning at early stages to collect centrosomal components. Immediately following prophase, cells begin assembling bipolar spindles. While the establishment of spindle bipolarity fails in the physical or functional absence of Eg5, I show that co-inhibition of dynein restores a cell’s ability to organize microtubules into a bipolar structure. Despite inhibition of both Eg5 and dynein, these spindles are morphologically and functionally equivalent to controls. Together, these data suggest that Eg5 and dynein share an antagonistic relationship and that a balance of forces, rather than a definitive set of players, is important for spindle assembly and function. To determine how Eg5- and dynein-mediated forces functionally coordinate to bring about antagonism during spindle assembly, I utilize a nocodazole washout assay. I show, via in vivo imaging and in silico modeling, that spindle collapse in the absence of functional Eg5 requires dynein activity and an initial intercentrosomal distance of less than 5.5μm. These data are consistent with a model in which dynein antagonizes Eg5 by crosslinking and sliding antiparallel microtubules, a novel role for dynein within the framework of spindle assembly.

Thesis advisor: David R. Burgess / Microtubule (MT) asters are radial arrays of MTs nucleated from a microtubule organizingcenter (MTOC) such as the centrosome. Within many cell types, which display highly diverse size and shape, MT asters orchestrate spatial positioning of organelles to ensure proper cellular function throughout the cell cycle and development. Therefore, asters have adopted a wide variety of sizes and morphologies, which are directly affects how they migrate and position within the cell. In large cells, for example during embryonic development, asters growth to sizes on the scales of hundreds of microns to millimeters. Due to this relatively enormous size scale, it is widely accepted that MT asters migrate primarily through pulling mechanisms driven by dynein located in the cytoplasm and/or the cell cortex. Moreover, prior to this dissertation, significant contributions from pushing forces as a result of aster growth and expansion against the cell cortex have not been detected in large cells. Here we have reinvestigated sperm aster growth, morphology, and positioning of MT asters using the large interphase sperm aster of the sea urchin zygote, which is historically a powerful system due to long range migration of the sperm aster to the geometric cell center following fertilization. First, through live-cell quantification of sperm aster growth and geometry, chemical manipulation of aster geometry, inhibition of dynein, and targeted chemical ablation, we show that the sperm aster migrates to the zygote center predominantly through a pushing-based mechanism that appears to largely independent of proposed pulling models. Second, we investigate the fundamental principles for how sperm aster size is determined during growth and centration. By physically manipulating egg size, we obtain samples of eggs displaying a wide range of diameters, all of which are at identical developmental stages. Using live-cell and fluorescence microscopy, we find strong preliminary evidence that aster diameter and migration rates show a direct, linear scaling to cell diameter. Finally, we hypothesize that a collective growth model for aster growth, or centrosome independent MT nucleation, may explain how the sperm aster of large sea urchin zygotes overcomes the proposed physical limitations of a pushing mechanism during large aster positioning. By applying two methods of super resolution microscopy, we find support for this collective growth model in the form of MT branching. Together, we present a model in which growth of astral MTs, potentially through a collective growth model, pushes the sperm aster to the zygote center. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

<p> The mode of chromosome duplication during meiosis and mitosis in Haplopappus gracilis was investigated. Tritiated thymidine was incorporated into the pollen mother cells during premeiotic interphase, and the cells were allowed to reach the tetrad stage. The autoradiographs prepared from the tetrads showed an unequal distribution of grains over their nuclei, suggesting a conservative mode of chromosome duplication during meiosis. Seedlings were fed with tritiated thymidine for the duration of one cell cycle and also for the duration of several cell cycles. The autoradiographs prepared from the root tip cells, thus treated, showed both labelled and unlabeled chromatids in the anaphases of all the experiments, thus again suggesting a conservative mode of chromosome duplication. A chromosome model to explain the results is discussed. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

No description available.

Equal segregation of the replicated genomic content to the two daughter cells is the major task of mitotic cells. The segregation is controlled by a complex system in the cell and relies mainly on the interaction between microtubules (MTs) of the mitotic spindle and kinetochores (KTs), specialized protein structures that assemble on each chromatid of each mitotic chromosome. By combining computational modeling and quantitative light microscopy, we established a quantitative model of the forces and regulators controlling metaphase chromosome movement in the mammalian cell line derived from Potorous tridactylis kidney epithelial cells (PtK1) (Chapter 2). This model can explain key features of metaphase chromosome dynamics and related chromosome structural changes experimentally observed. Moreover, the model made predictions, which we tested experimentally, on how changes in spindle dynamics affect certain aspects of chromosome structure. This quantitative model was next used to study the metaphase dynamics of chromosomes with erroneous KT-MT attachments (Chapter 3). Once again, the model predictions were tested experimentally and showed that erroneous KT-MT attachment alters the dynamics not only of the mis-attached KT, but also of its sister KT. Even more strikingly, experimental data showed that the presence of a single mis-attached KT could perturb the dynamics of all other, normally attached, KTs in anaphase. Chapter 3 also describe how MT poleward flux ensures correct KT-MT attachment and correct chromosome segregation. Indeed, reduced flux is associated with an increase in merotelically attached anaphase lagging chromosomes (LCs). These LCs form micronuclei (MNi) upon mitotic exit. The final effort of this work focused on the fate of MNi and micronuclated (MNed) cells (Chapter 4). Experimental observations showed that most of the chromosomes in MNi missegregated at the cell division following MN formation and that frequently the chromatin in the MN displayed delayed condensation. This work, thus, established a direct link between LCs and aneuploidy through the MN cell cycle. / Ph. D.

The degree of DNA supercoiling in the cell is carefully controlled by DNA topoisomerases. These enzymes catalyze the passage of individual DNA strands (Type I DNA topoisomerases), or double helices (Type II DNA topoisomerases) through one another. The purpose of the present study is to conduct a detailed analysis of the topo llα and β mRNAs expressed in several vertebrate cell lines. The final aim of this project is to analyze the relative roles of topo llα in chromatin condensation and chromosome segregation during mitosis, by performing topo llα gene targeting experiments in the DT 40 chicken lymphoblastoid cell line. The knock-out strategy was based on the observation of a high rate of homologous recombination versus random integration in the DT40 cell line. The topo llα gene was shown to be located on the chicken chromosome 2 (APM unpublished), for which the DT40 cell line is trisomic. The targeting vector completely replaced the 32 kb topo IIα genomic locus, generating a topo llα (-/+/+)cell line, which showed an increased resistance to topo II inhibitors. Paradoxically, 150 uM etoposide or 100 uM mitoxanthrone induced apoptosis within 5 hours in the topo llα (-1+1+) cell line, more rapidly as compared to the normal DT 40 cells. A topo IIα (-I-I+) cell line has also been generated. This study revealed the presence of evolutionarily conserved alternatively spliced forms of both topo llα and β isoforms between birds and humans. Hybridization screening of two chicken cDNA libraries, MSB-1 and DU249, revealed the presence of two distinct forms of both topo llα and β cDNAs. One form of topo llα, designated topo llα-1, encodes the chicken topo llα amino acid sequence previously reported (dbjiAB007445) in the database (unpublished). The second form, designated topo llα-2, encodes a protein containing an additional 35 amino acids inserted after Lysine-322 of chicken topo IIα-1 protein sequence. In the case of topo 11(3 mANA, one form, designated topo IIβ-1, encodes the protein already described (dbjiAB007446). The second form, tapa IIβ-2, would encode a protein missing the next 86 amino acids after Valine-25 in tapa II β-1 protein sequence. The tapa 11β variant is positioned similarly to one previously described in human (Hela) cells. The 5 amino acid insertion in the human tapa 11β-2 variant follows v23. In chicken cells, a longer insertion of 86 amino acids sequence follows v25, the homologous position in the tapa 11β protein. In human cells, the situation with tapa llα is more complex, as revealed by RT-PCR experiments (APM, unpublished) which generated several bands. One of these amplified species was found to contain a 36 amino acids insertion, positioned after residue K321 in the human tapa IIα cDNA, similarly to chicken tapa IIα-2 variant. The second human tapa llα spliced form cDNA was shown to contain a 26 amino acids insertion after residue A401 in the canonical human tapa llα protein sequence. The third cDNA variant isolated from human cells was described to encode a 81 amino acids insertion after residue Q355 positioned within the known human tapa IIα protein. It appears possible that the posttranslational modifications of the a-2 and β-2 isoforms may differ substantially from those of the canonical a-1 and β-1 isoforms. Such variant proteins could fulfil specialized functions, which might be tissue or cell-type specific. In summary, two novel forms of tapa llα and β cDNAs have been identified in three chicken cell lines. These spliced versions of both tapa llα and 13 isoforms seem to be evolutionary conserved, with similar forms occurring in their human counterparts. Future functional analysis of vertebrate tapa IIα and β will have to account for the existence of these novel isoforms, which might encode proteins that may exhibit different regulation of their subcellular localization, interaction with other proteins, or catalytic activity.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.