Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 100).

Proper orientation of the mitotic spindle is essential during animal development for the generation of cell diversity and organogenesis. To understand the molecular mechanisms regulating this process, genetic studies have implicated evolutionarily conserved proteins that function in diverse cell types to align the spindle along an intrinsic cellular polarity axis. This activity is achieved through physical contacts between astral microtubules of the spindle and a distinct domain of force generating proteins on the cell cortex. In this work, I shed light on how these proteins form distinct cortical domains, how their activity is coupled to their subcellular localization, and how they provide cytoskeletal and motor protein connections that are required to generate the forces necessary to position the mitotic spindle. I first discuss the mechanisms by which Mushroom body defect (Mud; NuMA in mammals), provides spindle orientation cues from various subcellular locations. Aside from its known role at the cortex as an adapter for the Dynein motor, I reveal novel isoform-dependent Mud functions at the spindle poles during assembly of the mitotic spindle and astral microtubules, thus implicating Mud in spindle orientation pathways away from the cell cortex. Moreover, through collaborative efforts with former lab members, I describe molecular regulation and assembly of two ‘accessory’ pathways that activate cortical Mud-Dynein, one through the tumor suppressor protein Discs large (Dlg), and another through the signaling protein Dishevelled (Dsh). I demonstrate that the Dlg pathway is spatially regulated by the polarity kinase atypical Protein Kinase C (aPKC) through direct phosphorylation of Dlg. This signal relieves Dlg autoinhibition to promote cortical recruitment of the Dlg-ligand Gukholder (Gukh), a novel microtubule-binding protein that provides an additional connection between astral microtubules and the cortex that is essential for activity of the Dlg pathway. Lastly, I determine that the Dsh accessory pathway provides an alternative cytoskeletal cue by recruiting Diaphanous (Dia), an actin nucleating protein. By demonstrating interchangeability between the two accessory pathways, we conclude that Mud-Dynein is activated by various cytoskeletal cues and that the mode of activation is cell-context dependent. This dissertation includes unpublished and previously published co-authored material. / 10000-01-01

Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint genes.

2-Methoxyestradiol (2ME) is an endogenous estrogen metabolite with antiproliferative and antiangiogenic properties. 2ME also plays an active role in the induction of apoptosis, especially in cancerous cells. These properties have been confirmed by various in vitro and in vivo studies and render 2ME a potential antitumor agent. The mechanism of action of 2ME, however, is not yet fully elucidated and it is believed that multiple mechanisms are involved that may be dependent on cell type. The aim of this study was to investigate the differential effects of 2ME on cell growth, morphology and spindle formation in the non-tumorigenic MCF-12A breast cell line and the tumorigenic MCF-7 breast cell line. In dose-dependent studies, cell growth was determined spectrophotometrically. Light microscopy was used to investigate the morphological changes induced by 2ME and its effect on spindle formation was investigated by means of indirect immunofluorescence. The estrogen receptor status of the MCF-12A cells was confirmed with immunocytochemistry. In order to investigate the effect of 2ME on the length of the cell cycle, cells were blocked in early S-phase with hydroxyurea, then allowed to continue through the cell cycle and mitotic indices determined at regular time intervals. Checkpoint kinase and Cdc2 kinase assays were used to determine the effect of 2ME on relevant cell cycle kinases. Although 2ME inhibited cell growth in both cell lines, the MCF-7 cells were inhibited from much lower concentrations and growth inhibition was more pronounced than in the MCF-12A cells. Treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent, or not as prominent in the MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells. Although the two cell lines differ in their estrogen receptor status, this could not explain the differential effects, for 2ME has a very low affinity for the estrogen receptor. 2ME had no effect on the length of the cell cycle, but blocked MCF-7 cells in mitosis. There were no significant alterations in the phosphorylation status of Cdc25C after 2ME treatment. However, Cdc2 activity was increased to a greater extend in the MCF-7 cells than in the MCF-12A cells. Therefore, it is suggested that exposure to 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest, which may result in the induction of apoptosis. The tumorigenic MCF-7 cells are especially sensitive to 2ME treatment compared to the normal MCF-12A cells. 2ME shows potential for the treatment of breast cancer. Selecting the concentration of 2ME that has maximum inhibitory effect on tumorigenic, but minimal effect on normal cells is crucial in its possible application as antitumor agent. Furthermore, research concerning the differential action mechanisms of 2ME is essential to create a better understanding regarding the treatment of cancer and may possibly contribute to the development and/or improvement of novel chemotherapeutic agents. / Dissertation (MSc (Physiology))--University of Pretoria, 2008. / Physiology / unrestricted

The thesis deals with the design and construction of headstocks of a turning centre. The aim is to design a spindle with an output of 20 kW and a maximum speed of 5000 1/min. The research part of the thesis is focused on the description of the basic parameters of the headstock and turning centres needed for the construction of the headstock and contains an analysis of the available spindles on the market. The next part of the thesis deals with the selection and calculation of cutting conditions and the necessary parameters. Analytical calculations are then verified using FEM analysis. At the end of the thesis, a 3D model was developed and headstock drawing documentation.

Diploma thesis deals with the concept of interchangeable straight spindle head type VA1-C, used for automatic clamping in 80 kW spindle head from production of TOS Kuřim. Thesis contains survey of interchangeable heads, analysis of the spindle housing, strenght analysis of the spindle with the safety calculations in critical sec-tions and analysis of the clamping mechanism together with the calculation of the camping force, which holds the tools in the spindle. Analysis of the camping mechanisms contains calculation of the contact stress between balls and housings. All results are shown in the graphs. In the last part of the thesis are described the construction elements using the model of the interchangeable head. Enclosed is the assembly drawing of the spindle head, bill of material, detail drawing of the spindle and also 3D model of the interchangeable spindle head.

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.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Mechanotransduction is fundamental to many sensory processes, including balance, hearing and motor co-ordination. However, for such an essential feature, the mechanism(s) that underlie it are poorly understood. The mechanotransducing stretch receptors that relay information on the tonicity and length of skeletal muscles have been well-defined, particularly at the gross anatomical level, in a wide variety of species, encompassing both vertebrates and invertebrates. To date, there exists a wealth of data describing them, anatomically, as well as good electrophysiological data from stretch receptors of some larger organisms. However, comparatively few studies have succeeded in identifying putative mechanotransducing molecules in such systems. Nonetheless, this class of sensory mechanotransducers perhaps offer the best means of identifying molecules that permit the stretch-sensitivity of such endings, revealing new information about the underlying mechanisms of stretch receptors, and mechanoreceptors more generally. However, a different approach is clearly needed; a theoretical approach, utilising mathematical modelling, offers a powerful means of pooling the current wealth of knowledge on the reported electrophysiological behaviour of muscle stretch receptors. This study, therefore, develops an extended theoretical model of a stretch receptor system in order to reproduce, in silico, the reported behaviour of both vertebrate and invertebrate stretch receptors, within the same modelling environment, thus enabling the first quantitative framework for comparing these data, and moreover, making predictions of the likely roles of specific molecular entities within a stretch receptor system. Subsequently, this study utilises a model in vivo system to test these theoretical predictions. The genetic toolbox of D. melanogaster offers a wide range of tools that are extremely suitable for identifying mechanotransducing molecules in stretch receptors. However, very little is currently known about such endings in this organism. This study, therefore, firstly characterises a putative stretch receptor organ in larval Drosophila, the dbd neuron, via a novel experimental approach. It is shown that this neuron exhibits known properties of stretch receptors, as previously observed in other, similar organs. Furthermore, these observations bear out the predictions of the mathematical model. Having defined the dbd neuron as a muscle stretch receptor, pharmacological and genetic assays in this system, combined with predictions from the mathematical model, identify a key role for the recently-discovered DmPiezo protein as an amiloride-sensitive, mechanically-gated sodium channel (MNaC) in dbd neurons, with TRPA1 also acting in this system in a supporting role. These data confirm the essential role of an MNaC in mechanosensory systems, but also supply important evidence that, whilst the electrophysiological mechanisms in stretch receptors are remarkably similar across taxa, different species likely employ various molecular mechanisms to achieve this.

The evolution of novel protein functions and protein families is a fundamental question within both evolutionary biology and biochemistry. While many gene families follow predictable patterns of molecular tinkering, many protein families exist with completely novel functions now essential. The guanylate kinase protein interaction domain (GKPID) of the membrane associated guanylate kinases (MAGUK) represents a model system for the study of protein evolution in which a protein scaffolding domain has evolved from a nucleotide kinase ancestor. Here we elucidate the ancient mechanisms by which these new functions evolved by combining ancestral protein reconstruction with in vitro and cell-biological molecular experiments. We found that the GKPID's capacity to serve as a mitotic spindle-orienting scaffold evolved by duplication and divergence of an ancient guanylate kinase enzyme before the divergence of animals and choanoflagellates. Re-introducing a single historical substitution into the ancestral guanylate kinase is sufficient to abolish the ancestral enzyme activity, confer the derived scaffolding function, and establish the capacity to mediate spindle orientation in cultured cells. This substitution appears to have revealed a latent protein-binding site, rather than constructing a novel interaction interface, apparently by altering the dynamics or conformational occupancy of a hinge region that determines whether the binding site is exposed or hidden. Three further substitutions also conveyed a measure of ligand specificity to phosphorylated Pins, which is necessary in metazoan spindle orientation pathways. These findings show how a small number of simple, ancient genetic changes caused the evolution of novel molecular functions crucial for the evolution of complex animals and laid the groundwork for an entirely new family of metazoan scaffolding proteins. This dissertation contains previously unpublished, co-authored material.