No description available.

No description available.

No description available.

No description available.

Mitosis is a highly regulated process by which a cell duplicates and distributes its chromosomal DNA into two identical daughter cells equally. Equal distribution of the chromosomes is crucial for accurate propagation of genetic information. This is essential for maintaining viability and preventing genomic instability that can potentially lead to cancer. In order to avoid unequal distribution of chromosomes, cells employ a surveillance mechanism called the spindle assembly checkpoint (SAC). The SAC is an inhibitory signalling network, which delays segregation of chromosomes, until they have stably attached to spindle microtubules through their multi-protein platforms, known as kinetochores. The main target of the SAC is the anaphase promoter complex/ cyclosome (APC/C), an E3 ubiquitin ligase. Specifically APC/C and its activator Cdc20 are inhibited by the main effector of the SAC, called the mitotic checkpoint complex (MCC). The MCC consists of Cdc20, Mad2, Mad3 and Bub3 (except S. pombe) proteins, which are recruited to the unattached kinetochores to promote MCC assembly. Once the chromosomes stably attach to the spindle, the SAC is turned off, MCC disassembles, and APC/CCdc20 is released from the inhibition. Activated APC/CCdc20 then targets its two main substrates, securin and cyclin B, for proteasomal degradation, and thereby triggers anaphase onset and mitotic exit. SAC signalling involves many protein components, whose activities are essentially regulated by direct protein-protein interactions and/ or post-translational modifications. One of these major modifications is phosphorylation, which is mediated by the SAC kinases such as Aurora B, Mps1 and Bub1. A number of studies have characterised SAC related substrates of Aurora B and Mps1 kinases in several model organisms. On the other hand, Bub1 kinase activity has been thought to play a key role in chromosome bi-orientation and more of an auxiliary role in SAC activation. The aim of this study is to investigate the importance of Bub1 kinase activity for SAC response in fission yeast Schizosaccharomyces pombe (S. pombe). SAC activation assays, using various degrees of spindle perturbation, have demonstrated that Bub1 kinase activity plays an important role in SAC maintenance. In order to examine the pathways downstream of Bub1, we set out to indicate Bub1 substrates which may be involved in SAC signalling. According to studies in various species, Cdc20 appears to be a prominent candidate, whose phosphorylation by Cdk1 and Bub1 kinases has been reported to regulate its mitotic activity. To investigate whether Cdc20 is phosphorylated by Bub1 in vitro, we purified recombinant S. pombe proteins from insect cells. Subsequent kinase assays identified Cdc20 as an in vitro substrate of Bub1, and the phosphorylated sites in Cdc20 were mapped by mass spectrometry. To address if this phospho-modification is involved in SAC regulation, phosphorylation mutants of Cdc20 were analysed in terms of their abilities to activate and silence SAC in vivo. Results show that phosphorylation of Cdc20 C-terminus promotes SAC maintenance in response to spindle damage. Furthermore, the mutations mimicking Bub1-mediated phosphorylation of Cdc20 Cterminus restore the SAC defects in the absence of Bub1 kinase activity. In addition, we purified S. pombe mitotic checkpoint complex (MCC) from insect cells, and analysed the interactions between its components (Cdc20, Mad2 and Mad3) by cross-linking mass spectrometry. Crystal structure of S.pombe MCC has been determined recently, which lacks Mad3 C-terminus and flexible C-terminal tail of Cdc20. Using an MCC with full length Mad3, we identified novel interactions between the C-terminal tails Mad3 and Cdc20, which are in close proximity to the identified Cdc20 phosphorylation sites. Briefly, in this study we confirm the previously known roles of Bub1 kinase activity (chromosome bi-orientation). Moreover, we propose a new pathway (in addition to the well-established H2A pathway) mediated by Cdc20, that may be important to maintain the SAC response.

Robust control mechanisms ensure faithful inheritance of an intact genome through the processes of mitosis and cytokinesis. Different populations of the cytoplasmic dynein motor defined by specific dynein adaptor complexes are required for the formation of a stable bipolar mitotic spindle. This study analysed how different dynein subcomplexes contribute to spindle formation and orientation. Various dynein subpopulations were identified by mass spectrometry. I have shown that the dynein light chain 1 (DYNLL1) directly interacts with the kinetochore localised Astrin-Kinastrin complex as well as the spindle microtubule associated complex formed by CHICA and HMMR. I have characterised both complexes and identified unique functions in chromosome alignment and mitotic spindle orientation, respectively. I have found that Kinastrin (C15orf23) is the major Astrin-interacting protein in mitotic cells and is required for Astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Fixed cell microscopy revealed that cells over-expressing or depleted of Kinastrin mislocalise Astrin. Additionally, depletion of the Astrin-Kinastrin complex delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset (Dunsch et al., 2011). Using immunoprecipitation and microtubule binding assays, I have shown that CHICA and HMMR interact with one another, and target to the spindle by a microtubule-binding site in the amino-terminal region of HMMR. CHICA interacts with DYNLL1 by a series of conserved TQT motifs in the carboxy-terminal region. Depletion of DYNLL1, CHICA or HMMR causes a slight increase in mitotic index but has little effect on spindle formation or checkpoint function. Fixed and live cell microscopy reveal, however, that the asymmetric distribution of cor tical dynein is lost and the spindle in these cells fails to orient correctly in relation to the culture surface (Dunsch et al., 2012). These findings presented here suggest that the Astrin-Kinastrin complex is required for normal spindle architecture and chromosome alignment, and that per turbations of this pathway result in delayed mitosis and non-physiological separase activation, whereas HMMR and CHICA act as par t of a dynein-DYNLL1 complex with a specific function defining or controlling spindle orientation.

No description available.

The centromere is a specialized chromatin domain that serves as the assembly site for the mitotic kinetochore structure, thereby playing a fundamental role in facilitating the maintenance of the genetic information. A histone H3 variant termed CENP-A is specifically found at all active centromeres. Beyond this, however, little is known about how and to which extent the chromatin environment of centromeres modulates and contributes towards centromere identity and function. Here, I have employed a novel Human Artificial Chromosome (HAC), the centromere of which can be targeted by fusions to the tet repressor, to characterize the chromatin environment underlying active kinetochores, as well as to specifically probe the role of this environment in the maintenance of kinetochore structure and function. My data demonstrate that centromeric chromatin resembles the downstream regions of actively transcribed genes. This includes the previously unrecognized presence of histone H3 nucleosomes methylated at lysine 36 within the chromatin underlying functional kinetochores. Targeted manipulation of this chromatin through tethering of a heterochromatin-seeding transcriptional repressor results in the inactivation of HAC kinetochore function concomitant with a hierarchical disassembly of the structure. Through an even more selective engineering of the HAC centromere chromatin, I have provided evidence supporting a critical role for nucleosomes dimethylated at lysine 4 on histone H3 in facilitating local transcription of the underlying DNA. Tethering of different chromatin-modifying activities into the HAC kinetochore collectively reveals a critical role for both, histone H3 dimethylated on lysine 4 and low-level, non-coding transcription in the maintenance of the CENP-A chromatin domain. On one hand, repression of centromeric transcription negatively correlates with the maintenance of CENP-A and ultimately results in the loss of kinetochore function. On the other hand, increasing kinetochore-associated RNA polymerase activity to within physiological levels for euchromatin is associated with rapid loss of CENP-A from the HAC centromere. Together, my data point towards the requirement for a delicate balance of transcriptional activity that is required to shape and maintain the chromatin environment of active centromeres.

The human pathogen Candida albicans is polymorphic, and its ability to switch growth forms is thought to play an important role in virulence. The primary research aim of this thesis was to understand the role the mitotic exit network plays in C. albicans with particular focus on the Tem1 GTPase protein. This aim was split into three specific goals; to study the role of Tem1 through the construction of a regulatable tem1 mutant, to understand the regulation of Tem1 through localisation and protein interaction studies, and to construct new molecular tools utilising the NAT1 positive selection marker in order to achieve two previous goals. In this thesis we demonstrated that TEM1 is an essential gene in C. albicans, and its essential function is signalled through the Cdc15 protein. Surprisingly, Tem1p depleted cells arrested as hyper-polarised filaments containing one or two nuclei and ultimately lost viability. These filaments formed from budding yeast cells, suggestive of a blockage late in the cell cycle. Ultimately the failure of these filaments to undergo cytokinesis was linked to a defect in septin ring dynamics and the formation of actomyosin ring. To understand the regulation of Tem1 we localised both the Tem1 and Lte1 proteins and found that Tem1 localised to spindle pole bodies in a cell-cycle dependent fashion by recruited at the onset of S phase. In contrast, the Lte1 homolog localised to the daughter cell cortex prior to release into the cytoplasm at the end of the cell cycle. A yeast 2-hybrid analysis of the MEN components demonstrated the potential of Bfa1/Bub2 and Tem1 to form a complex and the ability of Tem1 to homodimerise which may play a role in its self-activation. In order to carry out various aspects of this work we constructed a fully functional set of cassettes, including the constitutively active ENO1 promoter, V5-6xHIS epitope tag and various fluorescent protein genes fused to the NAT1 positive selection marker. When considered together, these results indicate that Tem1 is required for timely mitotic exit and cytokinesis in C. albicans, similar to S. cerevisiae, but the final output of the pathway must have diverged.

Accurate chromosome segregation is critical as unequal distribution of the genomic DNA results in impaired cell function or cell death. Kinetochores, the multi-protein structures assembled on centromeric DNA, drive chromosome segregation. Chromosome segregation is under supervision of mitotic spindle checkpoint. The mitotic spindle checkpoint is a surveillance mechanism ensuring that cells enter anaphase with all kinetochores properly attached to spindle microtubules and thereby preventing missegregation. Some checkpoint proteins are localised at kinetochore where they generate and enhance the checkpoint signal. Mps1 (Mph1 in S. pombe) and Aurora B (Ark1 in S. pombe) kinases are required for precise chromosome segregation and mitotic spindle checkpoint in fission yeast. In this study we investigate the roles of Mph1 and Ark1 in regulating the S. pombe kinetochore protein Spc7, which is the homologue of human Blinkin/KNL1. We demonstrated that both kinases target the N-terminus of Spc7. Loss of phosphorylation on the candidate phosphosites results in sensitivity to microtubule depolymerizing drugs indicating mitotic defects. As Blinkin has been proposed to be a docking platform for checkpoint proteins, we tested the possibility that Mph1 kinase is involved in kinetochore targeting of checkpoint proteins, Bub1 and Bub3. Our results demonstrate that Mph1-dependent phosphorylation of Spc7 at conserved MELT motifs is required for Bub1 and Bub3 kinetochore localisation. We were able to reconstitute the interaction between Spc7 and the Bub proteins in vitro demonstrating that the Spc7 phosphorylation is sufficient for Bub1 and Bub3 association with Spc7, most likely with Bub3 making the Spc7 contact. Mimicking phosphorylation at the MELT motifs leads to constitutive Bub1 localisation at kinetochores. We also showed that the N-terminus of Spc7 has microtubule binding activity regulated by Ark1 kinase. Mimicking phosphorylation at Ark1 sites results in reduced amount of recombinant Spc7 co-precipitating with microtubules in microtubule binding assays. Moreover, two stretches of basic residues, that contribute to Spc7 microtubule binding activity, have been mapped in the extreme Nterminus of Spc7. Spc7 also interacts with PP1 phosphatase, Dis2 in S. pombe, which is required for checkpoint silencing, but the mechanism of this interactions remains to be determined. These findings allow us to speculate on Spc7 role(s) in coupling microtubule binding with spindle checkpoint activation and silencing.