1 |
Identification of novel protein interactors of the SV40 large T antigen using the yeast two hybrid systemCotsiki, Marina January 2002 (has links)
No description available.
|
2 |
Control of the mitotic spindle by dynein light chain 1 complexesDunsch, Anja Katrin January 2013 (has links)
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.
|
3 |
'SynCheck' : new tools for dissecting Bub1 checkpoint functionsLeontiou, Ioanna January 2018 (has links)
The accurate segregation of DNA during cell division is essential for the viability of future cellular generations. Genetic material is packaged in the form of chromosomes during cell division, and chromosomes are segregated equally into two daughter cells. Chromosome mis-distribution leads to genetic disorders (e.g. Down's syndrome), aneuploidy and cancer. The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor (the Mitotic Checkpoint Complex-MCC). The MCC delays anaphase, thus giving cells time to fix attachment errors. Although the major checkpoint proteins were identified several years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the checkpoint signal. Yeast genetics and proteomics have revealed that kinetochores are highly complex molecular machines with almost 50 kinetochore components and ~10 components of the spindle checkpoint machinery. Such complexity makes the separation of error correction, kinetochore bi-orientation and microtubule attachment functions very challenging. To circumvent this complexity, a synthetic version of the spindle checkpoint (SynCheck), based on tetO array was engineered at an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes that combined targeting, initially of KNL1Spc7 with Mps1Mph1 and later of Bub1 (but not Mad1) with Mps1Mph1 fragments, was able to activate the spindle checkpoint and generate a robust arrest. The system is based on, soluble complexes, which were formed between KNL1Spc7 or Bub1 with Mps1Mph1. The synthetic checkpoint or 'Syncheck' is independent of localisation of the checkpoint components to the kinetochores, to spindle pole bodies (SPBs) and to nuclear pores. By using the synthetic tethering system a Mad1-Bub1 complex was identified for the first time in S.pombe. Bub1- Mad1 complex formation is crucial for checkpoint activation. Bub1-Mad1 gets phosphorylated itself and is thought to act as an assembly platform for MCC production and thereby generation of the "wait anaphase" signal. The ectopic tetO array is an important tool, not only for generating MCC formation and activating the spindle checkpoint, but also for providing a nice system for analysing in vivo protein-protein interactions. The ectopic array is capable of not only recruiting checkpoint components, but also recruiting them in a physiological manner (similar to the unattached kinetochores). For this reason it was decided to adopt this system to examine the role of the conserved Bub1TPR domain in the recruitment of other spindle checkpoint proteins. This work represents two novel functions for the S. pombe Bub1TPR domain. For the first time in S. pombe, both in vivo tethering and in vitro experiments with purified, recombinant proteins showed that the Bub1 has the ability to homodimerise and to form a complex with Mad3BubR1 through its TPR domain. These results revealed that complex formation of Bub1 with Mad3BubR1 is important for checkpoint signalling and that the highly conserved TPR domains in BubR1Mad3 and Bub1 have key roles to play in their interactions.
|
4 |
Centrosome and Mitotic Spindle Organization in Human CellsLawo, Steffen 10 January 2014 (has links)
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.
|
5 |
Centrosome and Mitotic Spindle Organization in Human CellsLawo, Steffen 10 January 2014 (has links)
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.
|
6 |
Genetic analysis of the amino terminus of spindle pole component spc110p /Nguyen, Thu Xuan Thi. January 2000 (has links)
Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 100).
|
7 |
Studying centrosome formation and the consequences of centrosome loss in Drosophila melanogasterBaumbach, Janina January 2014 (has links)
Centrioles are conserved microtubule-based structures that are required for the formation of two important cellular organelles, centrosomes and cilia. Centrosomes form the poles of the mitotic spindle and consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that has the ability to nucleate and organise microtubules. Centrosome defects are implicated into a variety of human diseases including cancer, microcephaly, and ciliopathies. Therefore it is of great interest to understand the mechanisms that lead to centrosome formation and the consequences that centrosome defects have in cells. I have analysed the roles of several centrosomal proteins in centrosome assembly in Drosophila. My results indicate that Sak/PLK4 is only required for the initial step of centriole duplication, but has no further role in recruitment of PCM. I show that two proteins important for PCM recruitment, Asterless (Asl) and Spd-2, are preferentially phosphorylated when they are integrated into the centrosome and I identified these phosphorylation sites using a phosphoproteomic screen. A phosphorylation site in Asl is specifically phosphorylated in mitosis, and the phosphorylation state of Spd-2 regulates its maintenance at the centrosome, suggesting that phosphorylation of PCM proteins is an important mechanism to ensure PCM assembly specifically at the centrosome and in mitosis. I have performed a global transcriptional analysis of flies lacking centrosomes or having extra centrosomes to investigate the effects of centrosomal defects on a cellular level. Surprisingly, my results indicate that centrosome defects per se do not dramatically alter cellular physiology. Finally, I demonstrate that in the absence of centrioles acentrosomal microtubule-organising centres (aMTOCs) are formed in an Asl- and Cnn-dependent fashion, and I show that these aMTOCs can contribute to spindle focusing in acentrosomal cells.
|
8 |
Role of mto2 in temporal and spatial regulation of cytoplasmic microtubule nucleation in Schizosaccharomyces pombeGroocock, Lynda M. January 2010 (has links)
The microtubule [MT] cytoskeleton of S. pombe is a highly dynamic network of filaments that facilitates intracellular transport, determines cell polarity and plays an essential role in chromosome separation during mitosis. In fission yeast, MTs are nucleated in a temporally and spatially regulated manner from sites called Microtubule Organising Centres [MTOCs], through the activity of both the g-tubulin complex [g-TuC] and the Mto1/2 complex. The Mto1/2 complex determines the localisation of the g-TuC at MTOCs, which change throughout the cell cycle. As cells enter mitosis the cytoplasmic array of MT bundles depolymerise. They are replaced by the intranuclear mitotic spindle and cytoplasmic spindle pole bodyderived astral MTs that in turn give way to the formation of the post-anaphase array. Although much is known about the properties of each type of MT array, the mechanism by which the timing of MT nucleation at different MTOCs is regulated over the cell cycle remains unclear. In the Mto1/2 complex, Mto1 is thought to provide the primary interaction with the g-TuC, and Mto2 functions by reinforcing this interaction. Due to the lack of structural information for the Mto1/2 complex, the molecular mechanism of Mto1/2- mediated assembly of the g-TuC at MTOCs is unknown. The aim of my study is to investigate the possibility that the Mto1/2 complex is able to promote g-TuC assembly by forming a direct template. In addition, I will attempt to determine the molecular role of Mto2 within the Mto1/2 complex and examine ways in which regulation of Mto2 may influence the function the Mto1/2 complex at specific MTOCs. As part of the investigation into the mechanism of Mto2 function, an in vitro analysis of recombinant protein demonstrated that in the absence of Mto1, purified Mto2 is able to self-interact as a tetramer. I have confirmed this interaction in vivo and have also shown that Mto2 forms a dimer as cells enter mitosis. However, in the context of an Mto1/2 complex the significance of the change in Mto2 oligomeric state remains unknown. Hydrodynamic analysis of a truncated form of the Mto1/2 complex suggests that it may form a heterotetramer, a hypothesis which is consistent with the equimolar levels of Mto2 and Mto1 protein within the cell. This information provides some structural insight as to how the Mto1/2 complex may interact with the g-TuC at MTOCs. Further analysis of the Mto1/2 complex revealed that in vivo, the Mto1-Mto2 interaction is disrupted during mitosis. This was found to correlate with the hyperphosphorylation of Mto2, which occurs as cells enter mitosis. Subsequently, an in vitro kinase assay demonstrated that phosphorylation of the Mto1/2 complex reduces the stability of the complex. Mass spectrometry techniques and sequence conservation were used to identify several phosphorylated residues within Mto2 and the ability of these mutants to bind to Mto1 was analysed in vivo and in vitro. In summary, in this study I have uncovered a mechanism which allows fission yeast cells to regulate the nucleation of cytoplasmic MT nucleation in a cell-cycle dependent manner, through a phosphorylation-dependent remodelling of the Mto1/2 complex.
|
9 |
The spindle pole body in Saccharomyces cerevisiae is a dynamic structure /Yoder, Tennessee Joplin. January 2003 (has links)
Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 30-37).
|
10 |
Analysis of the spindle pole component Spc110p /Sundberg, Holly. January 1996 (has links)
Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [69]-76).
|
Page generated in 0.0808 seconds