Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

During mitosis, accurate chromosome segregation is essential: gain or loss of genetic information can be detrimental to cell viability, or promote tumourigenesis. The mitotic checkpoint (also known as the spindle assembly checkpoint or SAC) ensures accurate chromosome segregation by delaying cell cycle progression until accuracy can be guaranteed. Mps1 is a protein kinase that is crucial for mitotic checkpoint signalling and also for proper chromosome alignment at metaphase. However, the precise role of Mps1’s catalytic activity is still unclear. Here, I present AZ3146, a novel small molecule inhibitor of Mps1. AZ3146 inhibits recombinant Mps1 in vitro with an IC50 of ~35 nM, and has low activity against a panel of 50 kinases, suggesting a reasonable degree of selectivity. As predicted for an Mps1 inhibitor, AZ1346 treatment led to spindle checkpoint malfunction in cells, accelerated mitotic timing, and perturbed the kinetochore localisation of the checkpoint effector Mad2. AZ3146 has a negative effect on cell viability, suggesting it leads to detrimental missegregations. Thus, the cellular effects of AZ3146 are consistent with Mps1 inhibition, and I was able to use the compound confidently as a tool to further probe the role of Mps1 activity in cells.Strikingly, levels of Mps1 increased at unattached kinetochores following inhibition of its kinase activity, suggesting Mps1’s kinetochore localisation is regulated by its own activity. A kinase-dead GFP-Mps1 fusion protein only accumulated at kinetochores in the absence of endogenous, active Mps1, implicating intra-molecular interactions in regulation of Mps1’s kinetochore localisation. I confirm a role for Mps1 in the mechanism of chromosome alignment, but in contrast to previous reports I did not detect a decrease in Aurora B activity following Mps1 inhibition. On the contrary, both Mps1’s phosphorylation status and its kinetochore localisation were affected by treatment with the Aurora B inhibitor ZM447439, placing Mps1 downstream of Aurora B. As an alternative explanation for the alignment defect in cells with reduced Mps1 activity, I found that levels of the plus-end directed kinesin Cenp-E were markedly decreased at unaligned kinetochores. I propose a model in which catalytically active Mps1’s auto-release from kinetochores simultaneously promotes both mitotic checkpoint signalling and chromosome alignment by facilitating Mad2 dimerisation and Cenp-E binding at unattached kinetochores.

Live cell imaging is a unique tool for cellular research with a wide variety of applications. By streaming digital microscopic images an investigator can observe the dynamic morphology of a cell, track cell movement on a surface, and measure quantities or localization patterns of fluorescently labeled proteins or molecules. Digital image sequences contain a vast amount of information in the form of visually detectable morphological changes in the cell. We designed computer programs that allow the manual identification of visible events in live cell digital image sequences [Davis et al. 2007]. Once identified, the data are analyzed using algorithms to calculate the yield of individual events per cell over the time course of image acquisition. The sequence of event data is also constructed into directed acyclic graphs and through the use of a subgraph isomorphism algorithm we are able to detect specified patterns of events originating from a single cell. Two projects in the field of cancer research are here discussed that describe and validate the application of the event analysis programs. In the first project, mitotic catastrophe (MC) research [Ianzini and Mackey, 1997; Ianzini and Mackey, 1998; reviewed by Ianzini and Mackey, 2007] is enhanced with the addition of live cell imaging to traditional laboratory experiments. The event analysis program is used to describe the yield of normal or abnormal divisions, fusions, and cell death, and to detect patterns of reductive division and depolyploidization in cells undergoing radiation-induced MC. Additionally, the biochemical and molecular data used in conjunction with live cell imaging data are presented to illustrate the usefulness of combining biology and engineering techniques to elucidate pathways involved in cell survival under different detrimental cell conditions. The results show that the timing of depolyploidization in MC cells correlates with increased multipolar divisions, up-regulation of meiosis-specific genes, and the production of mononucleated cell progeny. It was confirmed that mononucleated cells are produced from multipolar divisions and these cells are capable of resuming normal divisions [Ianzini et al., 2009]. The implications for the induction of meiosis as a mechanism of survival after radiation treatment are discussed. In the second project, the effects of long-term fluorescence excitation light exposure are examined through measurements of cell division and cell death. In the field of live cell imaging, probably the most modern and most widely utilized technique is fluorescence detection for intracellular organelles, proteins, and molecules. While the technologies required to label and detect fluorescent molecules in a cell are well developed, they are not idealized for long term measurements as both the probes and excitation light are toxic to the cells [Wang and Nixon, 1978; Bradley and Sharkey, 1977]. From the event analysis data it was determined that fluorescence excitation light is toxic to multiple cell lines observed as the reduction of normal cell division, induction of cell death, and apparent morphological aberrations.

<p>Meningioma represent more than one-third of all primary central nervous system (CNS) tumors,and it can be classified into three grades according to WHO (World Health Organization) in terms of clinical aggressiveness and risk of recurrence. A key component of meningioma grades is the mitotic count, which is defined as quantifying the number of cells in the process of dividing (i.e., undergoing mitosis) at a specific point in time. Currently, mitosis counting is done manually by a pathologist looking at 10 consecutive high-power fields (HPF) on a glass slide under a microscope, which is an extremely laborious and time-consuming process. The goal of this thesis is to investigate the use of computerized methods to automate the detection of mitotic nuclei with limited labeled data.We built computational methods to detect and quantify the histological features of mitotic cells on a whole slides image which mimic the exact process of pathologist workflow. Since we do not have enough training data from meningioma slide, we learned the mitotic cell features through public available breast cancer datasets, and predicted on meingioma slide for accuracy. We use either handcrafted features that capture certain morphological, statistical, or textural attributes of mitoses or features learned with convolutional neural networks (CNN). Hand crafted features are inspired by the domain knowledge, while the data-driven VGG16models tend to be domain agnostic and attempt to learn additional feature bases that cannot be represented through any of the handcrafted features. Our work on detection of mitotic cells shows 100% recall, 9% precision and 0.17 F1 score. The detection using VGG16performs with 71% recall, 73% precision, and 0.77 F1 score.Finally, this research of automated image analysis could drastically increase diagnostic efficiency and reduce inter-observer variability and errors in pathology diagnosis, which would allow fewer pathologists to serve more patients while maintaining diagnostic accuracy and precision. And all these methodologies will increasingly transform practice of pathology, allowing it to mature toward a quantitative science.</p>

A cell must build a bipolar mitotic spindle in order to faithfully segregate replicated DNA. To do so, multiple microtubule nucleation pathways are utilized to generate the robust spindle apparatus. TPX2, a microtubule binding protein, holds crucial roles in both the Ran-dependent and Augmin-dependent pathways where microtubules are nucleated near the chromosomes and from pre-existing microtubules. However, the exact role TPX2 plays in branching microtubules is less understood. Here, we explored the effect of truncating the essential TPX2 C-terminal 37 amino acids on Augmin localization and branching microtubule activity. First, we depleted LLC-Pk1 cells of the Augmin subunit HAUS6 and show that microtubule nucleation around the chromosomes following a nocodazole washout is strongly reduced leading to exaggerated kinetochore microtubule growth. Next, we depleted endogenous TPX2 in LLC-Pk1 cells harboring full length or truncated TPX2 bacterial artificial chromosome (BAC) DNA. Results show that TPX2 710 LAP cells have reduced Augmin localization on the spindle fibers, which correlates with reduced microtubule regrowth in the chromosomal region. In TPX2 710 LAP cells, regrowth was like Augmin depleted cells. Therefore, we provide evidence that the far C-terminus of TPX2 is required for branching microtubule nucleation and that kinetochore microtubule growth is Augmin-independent. In addition, we investigated cell cycle regulation of TPX2 by mutating the S738 phosphosite in the C-terminal motor interacting region. We utilized BAC recombineering to create phospho-mimetic and phospho-null mutants. In combination with plasmid DNA knockdown/rescue, overexpression and spindle assembly assays, we show that the phosphorylation of the C-terminal domain contributes to early mitotic events. LLC-Pk1 cells showed a significant increase in aberrant spindle morphology and reduced spindle stability in the presence of 738A and absence of endogenous TPX2. While rescue with the alanine mutant caused in an increase in multipolar spindles, overexpression resulted in a strong dominant negative monopolar phenotype. Therefore, S738 appears to contribute to mitotic force regulation during mitosis. In conclusion, the far C-terminus of TPX2 and its regulation play a role in the formation of a proper mitotic spindle.

The requirement of DNA synthesis for the release of Ɣ-radiation-induced mitotic inhibition in mammalian cells has been studied. Mammalian cells in which DNA synthesis had been inhibited by treatment with fluorodeoxyuridine (FUdR) were not released from radiation-induced mitotic inhibition until the FUdR block was removed. After removal of the block, mitotic figures reappeared, but only after a time equivalent to the usual mitotic delay caused by the particular radiation dose employed. This suggests that repair of the mitotic inhibition lesion can not proceed unless the pathway for DNA synthesis is intact. Further evidence for the requirement of DNA synthesis in the release of mitotic inhibition came from the observation of radiation-induced synthesis of DNA during G₂, a stage in the cell cycle normally not associated with such synthesis. / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24946号 / 生博第508号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 松本 智裕, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

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In LLC-Pk1 cells, and most cultured mammalian cells, cell division is highly regulated to achieve equal sized daughter cells. During this process, duplicated centrosomes separate and establish a bipolar array called the mitotic spindle. The mitotic spindle is responsible for aligning the chromosomes at the metaphase plate, and separating sister chromatids during anaphase. Spindle positioning and elongation are thought to be driven by the interaction between dynamic astral microtubules and cortical dynein. Extensive research has revealed that dynein is anchored to the cortex via the highly conserved NuMA/LGN/Gαi ternary complex in metaphase and the additional PIP/PIP2/NuMA, or 4.1G/R/NuMA, pathways during anaphase. Although substantial research has been conducted on the proteins involved with this process, it is unclear exactly how a cell is able to generate forces for spindle positioning and elongation. Here, I use photoactivation and FRAP techniques to investigate the role of the midzone during spindle elongation, and how cortical dynein is able to drive this process. I provide evidence that microtubule sliding in the midzone is not precisely coordinated with pole separation, however the two actions are interdependent. In addition, I demonstrate that cortical dynein dynamics are significantly enhanced during anaphase, most likely due to an increased length and stability of astral microtubules. I hypothesize that this increased turnover rate allows for rapid redistribution of dynein throughout the cortex to ensure proper spindle elongation.