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.

No description available.

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that mediate protein transport from late Golgi or trans-Golgi network (TGN) to vacuole via prevacuolar compartment (PVC) in plant cells. The N-terminus of a VSR is believed to be important for cargo binding while its transmembrane domain (TMD) and cytoplasmic tail (CT) are essential for its correct subcellular localization. In this study, I first developed and tested an expression system using transgenic tobacco BY-2 cells to produce truncated VSR proteins (VSRNT) lacking the TMD/CT into the cultured media. The secreted VSRs bind specifically to the vacuolar sorting determinants (VSDs) of known vacuolar proteins and such binding is calcium dependent in vitro. Thus, VSR cargo proteins are likely secreted into the cultured media along with the truncated VSRs, which enable the identification of various VSR cargo proteins from the cultured media of transgenic cells. I then identified these putative VSR cargo proteins through liquid-chromatography with tandem mass spectrometry (LC-MS/MS) and Fourier transform mass spectrometry (FT-MS) using transgenic Arabidopsis cell suspension cultures PSB-D expressing these truncated VSRs. Among the 17 unique proteins found in the cultured media of transgenic Arabidopsis PSB-D cell line expressing VSRNT, an Arabidopsis glycosyl hydrolase family 3 protein At5g10560 (GH3) was chosen for further study on VSR-cargo protein interaction. GFP-tagged GH3 fusion protein was found to co-localize with VSR-RFP marker protein in PVC, whereas GH3 was also shown to interact with a VSR protein BP-80. Loss-of-function analysis demonstrated that the GH3 contained a vacuolar sorting determinant (VSD) for PVC targeting. / Suen, Pui Kit. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 77-84). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cao Boyang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

The aim of this project was to investigate the effects of a novel mouse cytoplasmic dynein mutation; Abnormal rear leg (Arl). Cytoplasmic dynein is a microtubule (MT) based motor protein important for diverse cellular processes including Golgi maintenance and retrograde transport of organelles. Arl is a mouse point mutation in the heavy chain subunit of dynein (Dync1h1). Homozygous Dync1h1Arl/Arl die at embryonic day 10. Dync1h1Arl/+ heterozygotes have a normal life span, but exhibit abnormal gait and hindlimb clasping during tail suspension, typical of neuronal dysfunction. Protein purification from wildtype and heterozygous brain tissue showed increased MT binding in Dync1h1Arl/+ compared to wildtype. Delayed endosomal trafficking was observed in EGF stimulated Dync1h1Arl/+ mouse embryonic fibroblasts (MEFs) compared to wildtype, in both fixed cells and using live cell imaging. Similarly, a delay in the reassembly of the Golgi complex after disruption with a MT depolymerisation agent, nocodazole, was observed in Dync1h1Arl/+ MEFs compared to wildtype. In addition, the Golgi complex was observed as being structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons using transmission electron microscopy (TEM) compared to the wildtype. TEM also revealed that the mitochondria were structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons compared to wildtype, and O2 consumption assays investigating their function showed the Dync1h1Arl/+ mitochondria to have increased respiration rates compared to wildtype. Thus, these data highlight the Arl mouse as an invaluable model for studying the mechanism of dynein function and the subsequent outcomes when they are compromised.

Maintenance of the intracellular NADH/NAD+ redox poise is vital for energy generation in cells. Gram-positive bacteria, including the antibiotic-producing organism, Streptomyces coelicolor, have evolved a regulatory protein Rex that both senses this ratio and mediates an adaptive response to changes in it. Rex is a dimeric redox-sensitive transcriptional repressor. It is capable of binding to both NAD+ and NADH, although only NADH is an effector, causing dissociation of the protein from operator (ROP) sites. As NADH levels rise during oxygen limitation Rex dissociates from its target genes allowing expression, which helps to restore the NADH/NAD+ ratio. Microarray-based expression studies had suggested that Rex regulated only a small number of genes. In this work, however, ChIP-on-chip analyses revealed 38 genes that are potential regulon members. Analysis of the Rex binding sites in S. coelicolor revealed new insights into the mode of binding and show that Rex can bind with low affinity to incomplete half sites. This work also focused on characterising two key Rex targets, ndh and nuoA-N, that encode non-proton-translocating and proton translocating NADH dehydrogenases, respectively. Whereas nuoAN is not essential and was not expressed in liquid media, ndh was essential for growth. Depletion of NDH from growing cells led to the induction of Rex target genes confirming that ndh and Rex play key roles in maintaining redox homeostasis. Structure-based dissection of Rex, via a close homologue in Thermus aquaticus, identified a key interaction between the NADH- and DNAbinding domains of Rex. An R29-D203' salt-bridge, that traverses the NADH binding and DNA binding domains of Rex, appeared to stabilise the DNA-bound form of Rex, but is ‘broken' in the presence of NADH. In the NADH-bound form of Rex, D203 alternatively interacts with Y111, which in turn interacts with the nicotinamide ring of NADH. In order to assess the importance of individual subunits in the dimeric Rex, a single-chain derivative was constructed and the NADH binding and DNA binding domains individually disrupted.

Culturing C2C12 myoblasts and myotubes with a combination of LPS, TNF-α, IFN-γ and IL1β for 18 hours was used to determine the effects of endotoxic shock on possible causes of the dysregulation of glucose homeostasis associated with the syndrome. The in vitro model was confirmed by the significant production of NO in both myoblasts and myotubes following treatment. The treatment resulted in significantly different results between both myocyte preparations with regards to the regulation of glycogen synthesis. In the myoblasts, the treatment significantly increased myoblast glycogen synthesis, in a NO-independent manner, as seen by the inclusion of the NO synthase inhibitor L-NAME. This stimulation was unlikely to be due to a change in either GS or Phosphorylase activity. However it may have been caused by a significant increase in glucose transport induced by the treatment. This latter increase was also NO-independent, as well as not requiring reactive oxygen species. Insulin-induced myoblast protein synthesis was impaired by the treatment, which is likely due to an impairment of insulin-stimulated ERK1/2 phosphorylation. In the myotubes the case was different, as the treatment significantly reduced glycogen synthesis in a NO-dependent manner. This correlated with a NO-dependent increase in GS phosphorylation, indicating it was less active, however measurements of GS fractional activity failed to confirm this. Insulin stimulation of myotube glycogen synthesis was impaired by the treatment in a NO-independent manner, which may have involved an impairment of the insulin signal to ERK1/2. However the latter impairment was NO-dependent, suggesting other contributory mechanisms. Endotoxic treatment significantly increased myoblast protein content, but failed to do so in myotubes. This effect in the myoblasts may be explained by a significant increase in protein synthesis between 6 and 12 hours of treatment. None of the effects observed in the study were due to the treatment compromising cell viability.

Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation.

Moloney Murine Leukemia Virus (M-MLV) proviral DNA is transcriptionally silenced in mouse embryonic cells by a repressor complex containing tripartite-motif-containing 28 (Trim28). Trim28 depends on post-translational modifications, such as sumoylation and phosphorylation, and its interactions with several co-repressor proteins to regulate its repressive activity. YY1 is one such Trim28 co-repressor protein, recently found to tether the Trim28 silencing complex to the M-MLV promoter. Here, we investigated the biochemical interaction of Trim28 and YY1, and the role of sumoylation and phosphorylation of Trim28 in mediating M-MLV silencing. Experiments probing the binding of YY1 and Trim28 in vitro suggested that their interaction occurs indirectly. Mutational studies demonstrated that the RBCC domain of Trim28 is sufficient for interaction with YY1 while the acidic region 1 and zinc fingers of YY1 were necessary and sufficient for its interaction with Trim28. Additionally, we found that the K779 residue was critical for Trim28-mediated silencing of M-MLV in embryonic cells. The repressor complex that silences M-MLV is very large and likely consists of many protein subunits. A few proteins contained in the repressor complex have been identified, including Trim28, but the identity of most of the components forming the repressor complex are unknown. We detected a new form of the complex that is of even high molecular weight and likely contains additional associated cofactors. We reported an approach for purifying this larger repressor complex and identified new candidates for cofactors that may potentially function in the silencing of M-MLV. We also examined the regulation of sumoylation in embryonic cells. Sumoylation conjugation is a post-translational modification that affects a diverse range of processes and is important for embryo survival. Overall inhibition of the SUMO pathways results in embryonic lethality demonstrating the importance of the SUMO pathways for embryonic viability; however, our understanding of SUMO function in embryos at the cellular and molecular level is still greatly lacking. We demonstrated that SUMO1 cannot be overexpressed in embryonic carcinoma and embryonic stem cells and that SUMO1 overexpression is prevented at a post-transcriptional level. This occurred specifically for SUMO1 and not for SUMO2 overexpression. Furthermore, blocking conjugation or increasing the deconjugation of SUMO1 to substrates significantly improved SUMO1 overexpression. The results indicate that the overexpression of SUMO1 protein, in itself, is tolerated in embryonic cells, but the accumulation of substrate(s) modified by SUMO1 appears to be strongly prevented by an embryonic-specific post-transcriptional mechanism.

No description available.