The centrosome is the major microtubule organising centre of cells and serves as a centralised location for controlling many cellular processes. A critical component of the centrosome is the γ-tubulin ring complex (γ-TuRC) which is required for the nucleation of microtubules, correct formation of the mitotic spindle and hence progression of the cell cycle. NEDD1 (mouse: Nedd1), was recently discovered as a centrosomal protein which functions primarily in targeting the γ-TuRC to the centrosome and spindle. Given the fundamental role of the centrosome in mitosis and other processes, it is no surprise that this organelle is essential during mouse development. To examine the precise role of the centrosome during development, this study analysed the expression and localisation of Nedd1 during mouse embryogenesis. This revealed a dynamic localisation of Nedd1 and the centrosome during development, and provides further evidence for their critical role in development. To investigate the regulation of NEDD1, its expression during the cell cycle was analysed. It was found that phosphorylation is the primary method of NEDD1 regulation. Additionally, it was observed that Nedd1 levels decreased upon the entry of mouse embryonic fibroblasts into cell culture-induced senescence (an irreversible state of cell cycle arrest). This correlated with a loss of centrosomal integrity. Ablation of Nedd1 in healthy cells caused premature senescence and centrosome abnormalities, suggesting that Nedd1 and the centrosome may contribute to this senescence. NEDD1 is also important in the recruitment of the γ-TuRC to the centrosome, which is essential for correct centrosome biogenesis and function. This study identified a 62 amino acid region of NEDD1 that interacts with γ-tubulin and can abrogate its function. Key residues important for this interaction were also revealed. Additional interacting proteins of NEDD1 were also identified, and the chaperone TCP-1α was characterised in more detail and shown to regulate NEDD1. Given the currently known functions of NEDD1, it was expected to be important in development. Zebrafish were chosen as a model to study this because of their many advantages for developmental studies. A zebrafish homologue of NEDD1 was identified that displayed a similar localisation and function to mammalian NEDD1. Depletion of this protein caused lethality or phenotypic abnormalities which were most obvious in the central nervous system, depending on the extent of knockdown. This demonstrates that NEDD1 is critical for development, particularly in the nervous system. The results presented in this thesis contribute to the understanding of the function and regulation of NEDD1, and thus also the centrosome, and highlights the importance of this protein during development. Additionally, this study forms the foundation for further work on centrosomes, using NEDD1 as a marker for centrosomal dynamics and function. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457741 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 51-57).

Since Bortezomib®, a proteasome inhibitor, was approved by US FDA for the treatment of multiple myeloma in 2003, proteasome is recognized as one of the most promising targets for cancer therapeutics. The proteasomes play a critical role in regulating the level of cellular proteins and recycling damaged and misfolded proteins. Although the activity of the proteasome is essential for normal cells, it is especially critical for the proliferation and survival of cancer cells. In an attempt to develop effective and safe proteasome inhibitor-based anticancer drugs, the Lee laboratory created a chemical library by a hybrid approach using a 4-piperazinylquinoline scaffold and a sulfonyl phamarcophore. It is known that the chloroquine scaffold possesses a weak proteasome inhibition activity, and chloroquine itself preferentially kills malignant cells over non-cancer cells, alone or in combination with other therapeutics. To identify compounds with desirable anticancer activities, I have screened the aforementioned chemical library. The screening yielded several hits with substantial efficacy and selectivity against malignant cells. In this thesis, I describe the functional mechanism of VR23, one of the most promising compounds identified from my screening, as it kills cancer cells up to 17 fold more effectively than non-cancer cells. Molecular docking and substrate competition studies revealed that VR23 binds to the β2 peptide of the 20S proteasome catalytic subunit. The IC50 value of VR23 in inhibiting trypsin-like proteasome activity is 1.0 nM. VR23 is also substantially effective in inhibiting chymotrypsin-like proteasome activity (IC50, 50-100 nM). The inhibition of proteasome activity by VR23 led to the accumulation of ubiquitinated cyclin E at centrosomes. This, in turn, induces abnormal centrosome amplification by a de novo centrosome synthesis pathway in cancer cells, but not in non-cancer cells. The presence of multiple centrosomes in single cancer cells results in cell cycle arrest at prometaphase and, eventually, cell death by apoptosis. Thus, VR23 possesses a very desirable property as a safe anticancer drug.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18703号 / 生博第322号 / 新制||生||43(附属図書館) / 31636 / 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 豊島 文子, 教授 西田 栄介, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Pericentriolar satellites (PS) are electron dense granules surrounding the centrosome, the major microtubule-organizing centre in eukaryotic cells. In cycling cells the centrosome promotes spindle assembly and the faithful execution of mitosis. In non-cycling cells it is involved in forming the cilium, a plasma membrane-resident organelle, which mediates crucial signalling pathways in development and tissue homeostasis. PS are thought to contribute to centrosome formation, through the microtubule-dependent transport of centrosome components, and they are involved in ciliogenesis and stress response. Moreover, several proteins that localize to PS are mutated in human ciliopathies and neurodevelopmental disorders. The precise roles of PS in the various molecular pathways and diseases are however poorly understood, in part due to the limited knowledge of their composition. In the first part of my study I performed a comprehensive analysis of the pericentriolar satellite proteome. This was achieved by sucrose sedimentation of PS, combined with affinity purification of a key PS component, PCM1. To eliminate contamination by centrosomes, the PS proteome was determined from wild-type cells as well as from two cell lines genetically engineered to lack centrosomes. Mass spectrometry identified 170 PS components including most of the previously described PS proteins, confirming the validity of the approach. Having determined the proteomic composition of PS from DT40 cells, I then performed validation studies both in chicken and human cell lines. In the second part of my study, I aimed to use the list of PS proteins to uncover new biological roles for pericentriolar satellites. I devised two distinct approaches to gain functional insights. First, I generated a cell line lacking PCM1 as a tool to study the role(s) of PS and PS components. Second, I performed loss-of-function studies on a set of new PS proteins to determine their function(s) in maintaining the canonical PS distribution and in forming primary cilia.

Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive. In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.

Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive. In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.

Tumor initiation and progression is dependent on the acquisition and accumulation of multiple driver mutations that acti­vate and fuel oncogenic pathways and deactivate tumor suppressor networks. This complex continuum of non-stochastic genetic changes in accompaniment with error-prone mitoses largely explains why tumors are a mosaic of different cells. Contrary to the long-held notion that tumors are dominated by genetically-identical cells, tumors often contain many different subsets of cells that are remarkably diverse and distinct. The extent of this intratumor heterogeneity has bewildered cancer biologists’ and clinicians alike, as this partly illuminates why most cancer treatments fail. Unsurprisingly, there is no “wonder” drug yet available which can target all the different sub-populations including rare clones, and conquer the war on cancer. Breast tumors harbor ginormous extent of intratumoral heterogeneity, both within primary and metastatic lesions. This revelation essentially calls into question mega clinical endeavors such as the Human Genome Project that have sequenced a single biopsy from a large tumor mass thus precluding realization of the fact that a single tumor mass comprises of cells that present a variety of flavors in genotypic compositions. It is also becoming recognized that intratumor clonal heterogeneity underlies therapeutic resistance. Thus to comprehend the clinical behavior and therapeutic management of tumors, it is imperative to recognize and understand how intratumor heterogeneity arises. To this end, my research proposes to study two main features/cellular traits of tumors that can be quantitatively evaluated as “surrogates” to represent tumor heterogeneity at various stages of the disease: (a) centrosome amplification and clustering, and (b) mitotic frequency. This study aims at interrogating how a collaborative interplay of these “vehicles” support the tumor’s evolutionary agenda, and how we can glean prognostic and predictive information from an accurate determination of these cellular traits.

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.