タウオパチー変性神経細胞では、本来軸索に局在する微小管結合タンパク質タウが細胞体や樹状突起に蓄積するとともにチューブリン・微小管が減少する。この広範な神経細胞骨格系の変性は、タウの異常リン酸化に起因すると考えられてきた。しかし、タウの異常性獲得と微小管の消失との関係は未だ不明な状態にある。本研究では、初代培養神経細胞でαチューブリンの分子シャペロンであるTubulin-specific chaperon Eの発現抑制を行うことで、チューブリンの恒常性破綻を誘導するともに、それとタウの異常性獲得の関連性について検討した。 / Tauopathy is a type of neurodegenerative disorder including Alzheimer's disease defined by formation of tau filamentous inclusion in neurons. Tau is a microtubule associated protein localized in axon and assumed to promote microtubule stabilization in healthy neuron. In contrast, accumulation of hyperphosphorylated tau in somatodendrite and loss of microtubules (tubulin) are observed in tauopathy neuron. Although it is believed that abnormal phosphorylation of tau results in neurodegeneration, the relation with tau abnormalities and microtubule loss remains unclear. To investigate whether disruption of tubulin homeostasis induce tau abnormalities, we performed a miRNA-mediated knockdown of tubulin-specific chaperon E, an essential factor for the formation of alpha and beta tubulin heterodimeric complex, in mouse primary hippocampal neuron. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

No description available.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

We designed a new series of enediyne and their derivatives, and evaluating for their growth inhibition activity against human tumor cell lines. This dissertation is composed of two parts. First, compounds 6c, 6e, 6g and 7g displayed good growth inhibition activity against A549 (non-small-cell lung cancer), AGS (human stomach adenocarcinoma), PC-3 (prostate cancer), BT483 (breast carcinomas), HeLa (human cervical epithelioid carcinoma), OVCA (ovarian cancer cell line), SKHep (hepatocellular carcinoma), H460 (human lung cancer cell line) and SW620 (Human Colorectal Cancer Cell Line), especially compound 7g is better, and its average IC50 is 8.79£gM. Second, compounds 25a and 26 displayed better growth inhibition activity against HeLa (human cervical epithelioid carcinoma), OVCA (ovarian cancer cell line), AGS (human stomach adenocarcinoma) and H460 (human lung cancer cell line), and their average IC50 are10.82 and 11.08 £gM.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

No description available.

No description available.

Drosophila nonclaret disjunctional (Ncd) is a kinesin-like C-terminal motor protein that is involved in spindle assembly in oocytes during meiosis and in spindle maintenance in early embryos during mitosis. Ncd interacts with both "highway" and "cargo" microtubules (MTs) in meiotic and mitotic spindles through the action of ATP-dependent and ATP-independent MT binding sites in the head and tail domains, respectively. Through the action of these binding sites, Ncd bundles and, perhaps, slides MTs relative to each other. These functions are important for the in vivo role of Ncd in the formation of the bipolar spindle and maintenance of the spindle assembly. Despite the high homology of the Ncd head domain to the kinesin head domain, the Ncd tail domain is unique among kinesin-like motor proteins. Characterization of ATP-independent interactions of Ncd with cargo MTs and identification of MT binding sites (located in amino acid residues 83-100 and 115-187) in the tail region by MT co-sedimentation assays revealed that the Ncd tail has functional similarities to microtubule-associated proteins, especially to tau and MAP2, that regulate MT assembly. Like tau MT binding motifs, MT binding sites of the tail domain are rich in basic amino acids that are flanked by proline residues. Cross-linking and MT co-sedimentation assays with subtilisin-digested MTs demonstrated that Ncd tail binding sites (located at the extreme C-terminus and in the H11-H12 loop / H12 helix of each tubulin monomer) on tubulin correspond to tau binding sites. Further, the Ncd tail domain, like tau, can promote and stabilize MT assembly under conditions that induce MT disassembly. Taken together, these results suggest that the Ncd tail functions both in the transport of cargo MTs to spindle poles for the formation of the spindle assembly during meiosis, and in maintenance of spindle assembly during mitosis. How these different functions of Ncd are regulated still remains unknown, however further understanding of the regulation of Ncd function should contribute to our knowledge of cell cycle regulation in both meiotic and mitotic cells. / Ph. D.

Chemical modification is a powerful technique for probing functionally important amino acids. N-ethylmaleimide (NEM) reacts readily with exposed sulfhydryl groups, and has previously been shown to inhibit the activity of MT-motor proteins and tubulin assembly. This project seeks to investigate the mechanisms by which NEM affects motor function and inhibits MT minus end assembly. Recombinant motor domains of Drosophila kinesin (DK350 and DK375), Ncd (MC1), and squid kinesin (p181) were modified by NEM. NEM treatment was shown to affect the binding of MC1, but not recombinant kinesin proteins to MTs in the co-sedimentation assay. NEM treatment decreased the MT-stimulated ATPase rates of MC1 and DK350 in an NEM-concentration dependent manner, but did not affect the rate of DK375. Observed effects with DK375, p181, and MC1 were correlated with the number of labeled cysteines determined with [3H]NEM. As previously known, when NEM-treated tubulin was combined with untreated tubulin at certain ratios, assembly occurred only at the MT plus end. To investigate the mechanism by which NEM affects the polarity of tubulin assembly, tubulin was treated with NEM and assembly was analyzed using video-enhanced differential interference contrast microscopy. [3H]NEM was used to follow the time course of modification and to determine the number of modified sites per tubulin subunit. After 10 minutes, one cysteine was labeled on both a and b tubulin and this was sufficient to inhibit minus end assembly. Additionally, having one subunit labeled out of five tubulin subunits was sufficient to observe this effect. Protein digestion methods were used to aid in elimination of cysteines, to characterize potential critical cysteines in MC1, a, and b tubulin. / Master of Science

Microtubules (MTs) are important components of the eukaryotic cytoskeleton, with critical functions in intracellular trafficking, establishing and maintaining cell morphology, and segregating chromosomes during mitosis. MTs are hollow, cylindrical polymers composed of αβ-tubulin heterodimers. The longitudinal assembly of αβ-tubulin subunits generates protofilaments, and multiple protofilaments (typically 13 in vivo) interact laterally to form the wall of the MT. In vitro, the polymerization of MTs proceeds in two steps: nucleation and elongation. During the nucleation phase, several αβ-tubulin subunits associate to form a seed, from which further MT elongation then occurs. However, at the relatively low αβ-tubulin concentrations found in vivo, the spontaneous assembly of MTs is not favoured, due largely to the slow kinetics of MT nucleation. The nucleation of MTs in vivo requires the γ-tubulin complex (γ-TuC), a ring-like complex composed of γ-tubulin and γ-tubulin complex proteins (GCPs). Two copies of γ- tubulin associate with one copy each of GCP2 and GCP3 to produce the γ-tubulin small complex (γ-TuSC). Multiple γ-TuSCs, along with the additional GCPs 4,5, and 6, assemble to form the larger γ-tubulin ring complex (γ-TuRC). The γ-TuRC contains a ring of 13 γ-tubulins, which acts as a template for the nucleation of MTs. Typically, the γ-TuC nucleates MTs only when localized to specific subcellular sites, referred to as microtubule organizing centres (MTOCs). However, the precise mechanism by which the γ-TuC is activated at MTOCs remains unknown. In fission yeast, the proteins Mto1 and Mto2 form a complex (Mto1/2) required for the nucleation and organization of cytoplasmic MTs. Mto1/2 determines sites of MT nucleation by recruiting the γ-TuC to several different MTOCs. Different sequences in the Mto1 C-terminus independently confer γ-TuC localization to spindle pole bodies, MTs, and the cell equator. Here, I show that the Mto1 N-terminus is necessary for localization to the nuclear envelope (NE). By simultaneously removing the N- and C-terminal localization domains, I generated the "Mto1-bonsai" mutant, which fails to localize to any conventional MTOCs. In mto1-bonsai cells, MTs are still nucleated in the cytoplasm in an Mto1- dependent manner, but nucleation is spatially random. This reveals that targeting of the γ- TuC to conventional MTOCs is not necessary for MT nucleation, and suggests that Mto1/2 has a direct role in activating MT nucleation by the γ-TuC. Live-cell confocal microscopy allows us to detect individual MT nucleation events, in which newly nucleated MTs are associated with single γ-TuCs as well as Mto1/2-bonsai complexes. Fluorescence quantification reveals that these nucleating complexes contain approximately 13 molecules of both Mto1-bonsai and Mto2, matching the 13 copies of γ-tubulin anticipated for a single γ-TuC. We propose that Mto1/2 may contribute to γ-TuC activation by promoting γ-TuSC assembly and/or inducing conformational changes in the γ-TuC upon binding. I also expressed and purified recombinant Mto1/2-bonsai complex, using a baculovirus/insect cell system. This recombinant Mto1/2-bonsai self-assembles into higher-order complexes, comparable in size to the complexes analyzed in vivo by fluorescence microscopy.