No description available.

No description available.

The Hippo (Hpo) signalling pathway comprises the kinases Hpo and Warts (Wts), the adaptors Salvador and Mats, the cytoskeletal proteins Expanded (Ex) and Merlin (Mer), the atypical cadherin Fat and the transcriptional co-factor Yorkie (Yki). This pathway has been shown to restrict tissue size through the control of cell division and apoptosis during development in Drosophila. In addition to their well-characterised overproliferation phenotype, adult epithelial cells mutant for the kinases Hpo and Wts present a hypertrophy of the apical domain. I examined the molecular basis of this apical hypertrophy and its impact on cell proliferation. In the wing imaginal disc epithelium, I observe increased staining for members of the apical polarity complexes aPKC and Crumbs as well as adherens junction components when Hpo activity is compromised, while baso-lateral markers are not affected. This increase in apical proteins is correlated with a hypertrophy of the apical domain and adherens junctions. Interestingly however, while the polarity determinant Crumbs is required for the accumulation of apical proteins, this does not appear to significantly contribute to the overproliferation defect elicited by loss of Hpo signalling. Therefore, the Hpo pathway controls growth and apical domain size via distinct mechanisms. In collaboration with the Thompson lab (CRUK LRI) we identified the WWdomain- containing protein Kibra (Kib) as a new member of the Hpo pathway. Kib, which colocalises and physically interacts with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kib/Mer interaction is conserved in human cells. Loss of kib induces a hpo-like phenotype and genetic experiments place it upstream of the core kinase cassette. Finally, Kib binds to Wts and kib depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. My work therefore suggests that Kib is part of an apical scaffold that promotes Hpo pathway activity.

Intercellular communication has been investigated between embryonic Xenopus neurones and myocytes in vitro and during digit patterning in the Chick wing bud. Lucifer yellow and Huridine were used to identify intercellular transfer and metabolic cooperation in both systems, and communication inhibition by a specific antibody raised against gap junctional protein confirmed the nature of this transfer. The molecular tracers and immunogenic reagents were loaded into tissues in bulk using DMSO permeabilisation. Dye transfer from embryonic Xenopus myocytes to neurones was observed during coculture. 24 hours after plating 77% of the loaded myocytes and 23% of the recipient neurones contained dye. The presence of gap junctional antibody in either cell type reduced the neuronal label to 4%, indicating cell coupling occurred. Metabolic cooperation experiments using the transfer of HNucleotide in both directions between the neurone and myocyte confirmed these findings. Blocking these gap junctions had no significant effect on neuronally induced AChR clustering on the myocytes. The signalling of the chick limb polarising region (PR) was studied by loading pellets of PR diluted with anterior mesenchyme (AM) with gap junctional antibody prior to grafting the tissue into the anterior margin of the host limb bud, and observing the resulting duplications. When PR cells alone were inhibited from intercellular communication more respecification of the host AM occurred, with greater duplications. In contrast, if both the diluting AM cells and the PR cells were loaded with antibody, reduced levels of duplication were obtained. Metabolic cooperation between dissociated PR and AM cells in culture confirmed that the antibody was active throughout the respecification period, and blocked gap junctions in the chick. Possible roles of intercellular communication in both systems are discussed. At the developing synapse gap junctions are probably not involved in the local structural rearrangements but may enable the neurones to recognise suitable targets for innervation in vivo. The chick results suggest that morphogenetic patterning in the limb bud may be more complex than previously recognised.

No description available.

One of the biggest challenges currently facing ES cell biology is to understand the mechanisms involved in the differentiation of ES cells to specific lineages. Pure populations of a specific cell lineage cannot be achieved without selection, such as fluorescence activated cell sorting (FACS), immunopanning or growing cells in a selective environment following genetic manipulation. However, such techniques do not address why ES cells do and do not differentiate to particular lineages and cell types. To achieve greater understanding of the mechanism of neuronal differentiation from ES cells, an ES cell line was generated with <i>eGFP </i>driven by the neuronal specific gene <i>tau. </i>Tau is expressed exclusively in all neurons from the earliest stages of neuronal commitment, we find that a neural differentiation of this line results in eGFP expressing neurons. Using FACS, a pure population of neurons can be obtained from a heterogeneous population of differentiated cells. Neuronal differentiation can be quantified either by fluorescent microscopy or flow cytometry. These ES cells have been used to analyse the effect that density and the addition of exogenous factors have on neuronal differentiation, a transcriptome analysis experiment was performed by microarray analysis. Genes already known to be important during mammalian neural development were analysed for their involvement in ES cell neurogenesis. This comparison revealed a strong correlation between events of ES cells differentiation and normal embryonic development. The microarray analysis of ES cell neurogenesis also identified genes with an expression profile suggestive of a role in ES cell neurogenesis and development of the murine nervous system.

The thesis discusses protein targeting within cells, and in particular targeting to the mitochondrion. The synthesis and conformational analysis by CD and NMR of a mitochondrial targeting presequence peptide, and a non-functional deletion peptide are described. The intramitochondrial sorting of proteins is also discussed and investigated. Ch. 1 reviews the currently available knowledge of the mechanisms and factors involved in protein targeting. Ch. 2 describes the synthesis and purification of the 25 residue peptide corresponding to the mitochondrial targeting presequence of subunit IV of yeast cytochrome oxidase. Ch. 3 describes the conformational analysis by CD and NMR studies of the synthetic peptide and discusses the structural information that can be drawn from the data. Both sets of data indicate that the peptide appears to adopt a partially helical structure. Ch. 4 describes the synthesis and purification of a 23 residue peptide corresponding to a non-functional deletion mutation of the cytochrome oxidase subunit IV targeting peptide. The CD and NNR studies carried out on this peptide, the preliminary results of which indicate an absence of any regular structural motifs, are discussed. Ch. 5 reviews the current understanding of the mechanisms of intramitochondrial sorting and their evolutionary relationship to protein secretion. E.Coli cells expressing cytochrome b2 with various portions of the presequence removed were subfractioned to determine whether any form of the presequence showed a strong tendency to target the protein to the cell membranes. Conclusive results were not obtained due to the formation of inclusion bodies within the cells.

The p53, Rb and Ras/PI3K pathways are implicated in the development of the majority of human cancers. A number of studies have established that these pathways co-operate at the level of the cell cycle leading to loss of normal proliferative controls. For this thesis I have investigated how these signals influence a second critical component of tumour formation – cell growth. I have discovered that while oncogenic Ras is sufficient to drive growth via the canonical growth pathway, PI3K–AKT–TOR, it does so relatively weakly and p53 loss does not drive cell growth at all. Importantly, this work identifies a novel role for the Rb family of tumour suppressors in directing cell growth via a signalling pathway distinct from PI3K–AKT–TOR and via an E2F-independent mechanism. However, I have found that strong, sustained growth requires Rb loss together with Ras signalling, identifying an additional mechanism by which these oncogenic pathways co-operate and a critical role for Ras in preserving the uptake of extracellular nutrients required for biogenesis.

The aim of this project is to identify and characterise novel client proteins of Cdc37 in <i>S. pombe</i> through a synthetic lethal genetic screen. During the screen, 68 strains were initially picked out of 40,000 colonies as potentially synthetically lethal with <i>cdc37-681</i>, a temperature sensitive allele, at permissive temperature. When retested, 12 strains were identified as candidate strains. Mutations identified in 3 strains were found by genetic analysis to have a phenotype of their own in a <i>cdc37⁺</i> background. A genomic library was transformed into each synthetic lethal mutant. <i>wis4, msc1, nak1 </i>and <i>cdc7</i> were identified as candidate genes that rescue the various mutants. <i>wis4</i> encodes a MAP3K involved in the stress-responsive signal transduction pathway; <i>win1</i> encodes a closely related kinase. By crossing <i>wis4</i> and <i>win1</i> deletion strains with <i>cdc37-681,</i> it was proved that <i>wis4</i> and <i>win1 </i>are not synthetic lethal with <i>cdc37-681. msc1</i> is a multi-copy suppressor of <i>chk1</i> and has a role in regulating chromatin structure. However, neither <i>musc1</i> nor <i>chk1</i> is synthetically lethal with <i>cdc37-681</i>. <i>nak1</i> encodes an essential protein kinase and plays a role in the regulation of cell polarity, growth and division. But <i>nak1ts</i> mutants do not show synthetic lethality with <i>cdc37-681</i>. Cdc7 is a protein kinase essential for septation and cell division.  A known <i>cdc7ts</i> mutant, <i>cdc7-24</i> was shown to by synthetically lethal with <i>cdc17-68.</i> The synthetic lethal mutation outcrossed from J322 (the strain rescued by <i>cdc7⁺)</i> shows the same phenotype as <i>cdc7-24</i>. Cdc7 is a possible Cdc37 client, but Cdc7 protein levels do not change in <i>cdc37-681 </i>or <i>cdc37-184</i> at restrictive temperature. Cdc7 kinase activity is produced in <i>cdc37-681</i> and <i>cdc37-184</i>, indicating that Cdc37 function is needed for Cdc7 kinase activity. The cell morphology of <i>cdc37ts</i> in combination with GFP-tagged <i>cdc7</i> is similar to that of <i>cdc7ts</i> mutants and differs from the <i>cdc37ts</i> single mutant. Cdc7 can locate to the spindle pole body in <i>cdc37ts</i> strains, which suggests Cdc7 localisation does not require Cdc37.

CAD (Caspase Activated DNase) exists in a complex with its inhibitor ICAD in intact cells. To study the particular role of ICAD splice forms ICAD-S and ICAD-L <i>in vivo</i> I constructed DT40 cell lines in which the ICAD or ICAD plus CAD ORFs had been deleted. ICAD and ICAD/CAD double knockouts were characterized by the absence of DNA fragmentation and apoptotic bodies after the induction of apoptosis. To characterise the functions of ICAD-S, I constructed a model system consisting from human (h) hICAD-S, hICAD-L and hCAD components. hICAD-S was not able to work as a chaperone for hCAD <i>in vivo</i>. However, a modified version of hICAD-S hICAD-S^2TEV was able to inhibit hCAD activation upon induction of apoptosis <i>in vivo. </i> Moreover, such inhibition correlated with level of hICAD-S^2TEV expression. The inhibitor function of hICAD-S^2TEV was confirmed <i>in vitro,</i> where hCAD was activated after hICAD-S^2TEV cleavage with TEV protease. The use of CAD as a drug target could be particularly advantageous since CAD is situated at the end of apoptotic pathway. Thus, once activated CAD may avoid upstream regulatory mechanisms that protect cells against certain apoptotic stimuli. I constructed a model system based on ICAD/CAD double knockout to study the possibility that CAD can cause the cell death. This system included hICAD-L^2TEV:hCAD and TEV protease under the control of inducible promoter. However, it was not possible to cleave hICAD-L^2TEV protein <i>in vivo</i> with TEV protease. An alternative strategy was based on the use of PreScission protease. The active part of PreScission protease is 3C protease from human rhinovirus origin. A cleaved fragment of hICAD-L^2PREprotein was detected in DT40 cells expressing PreScission protease. Preliminary data suggest that CAD contributes to cell death in cells expressing hICAD-L^2PRE:hCAD and transfected with PreScission protease.