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Bibliography: p. 115-132 / iii, 132 p. : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Concerns the characterisation of a gene, three throws, whose mutant phenotype of failure of chromosome disjunction in anaphase, is indicative of an essential but unknown function in mitosis. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1995

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Thesis (Ph. D.)--Lehigh University, 2003. / Includes vita. Includes bibliographical references (leaves 84-107).

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1951. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 43-52).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1948. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 74-82).

Chapter I. After a period of culture at low temperature, mitotic chromosomes of the newt species Triturus cristatus and T. vulgaris show a characteristic pattern of predominantly pericentrically located secondary constrictions. The chromatin in these constricted regions stains intensely with Giemsa. When mitotic chromosome preparations are stained according to a C-banding technique, the centromeres and the interstitial regions which stain differentially after cold-treatment, stain intensely with Giemsa. Electron micrographs of sections through metaphase chromosomes in tail-fin cells of cold- and colchicine-treated larvae show that the chromatin fibres are more densely packed in the constricted regions than elsewhere. Hypotonically treated spermatogonia or tissue-culture cells of T.c. carnifex show spiral structure throughout the metaphase chromatids. Chapter II. T.c. carnifex skin fibroblasts can be maintained in monolayer culture in a predominantly diploid state for more than 14 months. The cells grow at 25°C in Eagles' MEM supplemented with 10% foetal calf serum and glutamine. The cell generation time is approximately 4 days. This is the only diploid urodele cell line maintained in any laboratory. Chapter III. Purified and iodine-labelled ribosomal RNA extracted from T.c. carnifex ovaries hybridises in situ to a region 2/5 of the way down the long arms of both chromosomes X of T.c. carnifex tissue culture cells. When this RNA preparation is hybridised in situ to mitotic chromosomes of T.c. carnifex larval brain cells, labelled regions are found (i) near the telomeres of both chromosomes II, (ii) halfway down the long arms and at the ends of the long arms of both chromosomes X, (iii) near the centromere of one large metacentric chromosome, and (iv) halfway down the long arm of a medium-sized submetacentric chromosome. There is a variation in the labelling pattern shown by different T. vulgaris animals, and perhaps some cell to cell variation with the same animal. Mitotic metaphase chromosomes of T.c. carnifex spermatogonia hybridised in situ with iodine-labelled 5s RNA are labelled about halfway down the long arms of both chromosomes X. The numbers of nucleoli in methyl green/pyronine or "silver stained" cells of T.c. carnifex tissue culture or spermatogonia and T. vulgaris larval brain, are greater (up to 6-fold) than expected from in situ hybridization results. The apparent increase in nucleolar number is probably due to fragmentation of material from the original nucleoli. Chapter IV. The sister chromatid exchange frequence of T.c. carnifex tissue culture cells analysed by the FPG technique increases from about 20 at 1 mug/ml BUdR to about 50 at 100 mug/ml BUdR.

The experimental component of this thesis represents the continuation of studies carried out in the Milner laboratory to characterise the biology of Drosophila imaginal disc cell lines growing in vitro. The bulk of this study was concerned with the morphological and molecular characterisation of imaginal cell interactions in vitro and contrasting this with what is known of imaginal cell biology in vivo. The imaginal cell lines were thus seen as both an amenable model for the detailed analysis of a cells interaction with its environment and, more broadly, as an assessment of the effects of isolating and maintaining animal cells in vitro. The reaggregation of single imaginal cells when in suspension is rapid and extensive when in the presence of the divalent cations, Ca2+ and Mg2+, but is not entirely dependent on them suggesting the existence of separate systems of adhesion and the presence of a variety of cell adhesion molecules (CAMs). During the course of growth in vitro there appears to be a shift in strategy between cell-substrate and cell-cell adhesion as cells move from monolayers into aggregates. A new set of leg cell lines were cloned, some of which had a radically reduced capacity to reaggregate in suspension suggesting that CAMs can be selectively lost from the imaginal cell surface. PS integrins are widely expressed in imaginal cells in vitro, as in vivo, and seem to be similarly involved in various cell adhesion events here. PS integrins are expressed at all stages of imaginal cell growth and function both in mediating cell-substrate adhesion, notably to human fibronectin, and in cell-cell adhesion. PS integrins appear to mediate a 'higher order' cell-cell adhesion via an unidentified component of the extracellular matrix (ECM) which again can be substituted using vertebrate molecules. PS integrins may direct aggregation by forming tension transmitting junctions with other cells, evidence for which is provided by F-actin and tubulin expression in aggregating cells. The in vivo epithelial phenotype, characterised by apical-basal polarity, can be re-established in part by growing cells on fibronectin-coated membranes in the presence of unidirectional nutrient uptake and a feeder layer of cells. Cells in vitro lack such polarity signified by the absence of specialised intercellular junctions and a loss of restriction of expression of the putative CAM and signalling molecule, CRUMBS. Another CAM which appears to be expressed during imaginal cell growth in vitro is the immunoglobulin, fasciclin III, or a variant, which is restricted in expression to single migratory cells and during cell-cell adhesion. Some evidence is also provided that these cell lines express, and can endocytose, proteins known as larval serum proteins'. The nature of this uptake remains obscure, but does not seem to be enhanced by the presence of the moulting hormone, 20-hydroxyecdysone (20-HE).

Upon entry into G2 and mitosis (G2/M), dynein dissociates from its interphase cargos and forms mitotic-specific interactions that direct dynein to the nuclear envelope, cell-cortex, kinetochores, and spindle poles to ensure equal segregation of genetic material to the two daughter cells. Although the need for precise regulation of dynein’s activity during mitosis is clear, questions remain about the mechanisms that govern the cell-cycle dependent dynein interactions. Frequently dynein cofactors provide platforms for regulating dynein activity either by directing dynein to specific sites of action or by tuning the motor activity of the dynein motor. In particular the dynein cofactor Nde1 may play a key role in defining dynein’s mitotic activity. During interphase, Nde1 is involved in the dynein-dependent processes of Golgi positioning and minus-end directed lysosome transport (Lam et al., 2009; Yi et al., 2011), but as the cell progresses into G2/M, Nde1 adopts mitotic specific interactions at the nuclear envelope and kinetochores. It is unknown how Nde1’s cell-cycle specific localization is regulated and how, if at all, Nde1 is ultimately able to influence dynein’s recruitment and activity at each of these sites. One candidate is cell-cycle specific phosphorylation of Nde1 by a G2/mitotic specific kinase, cyclinB/Cdk1 (Alkurayaet al. 2011). To study the potential function of the phosphorylation by Cdk1, we assayed the localization of GFP Cdk1Nde1 phospho-mimetic and phospho-mutant constructs at the NE and kinetochores. We demonstrate Cdk1 phosphorylation of Nde1 is required for Nde1 localization to both the NE and to the kinetochore, and also the phosphorylation of Nde1 directly activates physical interactions between Nde1 and its nuclear envelope and the kinetochore-binding partner, CENP-F. Furthermore, physiological studies of Nde1 phosphorylation constructs show that over-expression of GFP Nde1 phospho-mutant causes a significant delay in time from NEBD to anaphase onset, specifically demonstrating a late prometaphase/metaphase arrest. Therefore, we conclude Cdk1 phosphorylation of Nde1 not only regulates its localization to the nuclear envelope and kinetochore but also plays an important functional role in Nde1’s mitotic activity in vivo. In addition to understanding how the cell cycle specific activity of Nde1 is regulated, to fully comprehend how dynein functions during mitosis it is necessary to understand how Nde1 is able to modulate dynein’s activity. Nde1 is typically believed to act as a bridge between dynein and specific cellular cargo by physically interacting both with the cargo and dynein/Lis1 to specify the sites of dynein’s activity. Therefore, to understand how Nde1 functions with Lis1 and dynein during mitosis, we created point mutations in the N-terminal coiled-coil domain that specifically disrupted either the Nde1-Lis1 interaction or the Nde1-dynein interaction. We find that disrupting the Nde1-dynein interaction has more severe phenotypic effects compared to disrupting the Nde1-Lis1 interaction: expression of GFP Nde1 del dynein mutant caused a significant delay in anaphase onset while GFP Nde1 del Lis1 only caused a slight increase in cell cycle duration before anaphase onset. Phenotypic analysis suggests that the effects of abolishing the Nde1-dynein interaction on mitotic progression may be due to defects in maintaining kinetochore-microtubule stability during metaphase. Nde1 plays a role in this dynein-dependent mitotic activity through recruitment of a subfraction of dynein to the kinetochore by Nde1’s coiled-coil domain. While the phenotypic effect of removing the Lis1-Nde1 interaction is less severe than removing the dynein-Nde1 interaction, the interaction between Lis1 and Nde1 plays an important role in Nde1’s mitotic behavior as it is affects Nde1’s localization at the kinetochore, specifically by influencing Nde’1 interaction with its kinetochore recruitment partner, CENP-F. The entirety of this work demonstrates that Nde1 acts as a link between cellular cargo and dynein behavior as phospho-regulation of Nde1 throughout the cell cycle allows Nde1’s to interact with unique mitotic cargoes and influence the recruitment and activity of dynein at the kinetochore.