The small GTPase RHO-1 is an important regulator of neurotransmission. Caenorhabditis elegans nematodes expressing activated RHO-1 (G14V) in their cholinergic motor neurons (nRHO-1*) become hypersensitive to the acetylcholinesterase inhibitor aldicarb, demonstrating increased acetylcholine release, and acquire a highly loopy, uncoordinated locomotion. RHO-1 inhibits diacylglycerol kinase (DGK-1), and so increases the availability of diacylglycerol (DAG), a key second messenger for release at the presynaptic membrane. Inhibiting RHO-1 in a dgk-1 mutant causes a decrease in neurotransmitter release, demonstrating the presence of additional targets downstream of RHO-1. During a forward genetic screen for suppressors of the loopy locomotion of nRHO-1* animals we obtained a mutant, nz94, which carried an additional ‘fainter’ phenotype, helping us identify it as an allele of unc-80, a large, conserved protein, important in the localization of NCA-1 and NCA-2, C. elegans homologues of the novel mammalian ion channel NALCN. RHO-1*;unc-80 double mutants are non-loopy, but still hypersensitive to aldicarb, indicating that the loopy locomotion and high levels of neurotransmitter release can be uncoupled. unc-80 mutants do not suppress non-neuronal phenotypes associated with heat-shock expression of RHO-1*, such as tail swelling and sterility. Expressing an unc-80 transgene under a cholinergic promoter is sufficient to rescue the suppression of loopy locomotion seen in the nRHO-1*;unc-80 double mutants, indicating that unc-80 acts presynaptically in the same cells as nRHO-1* for the generation of this loopy locomotion. Work from other labs shows that unc-80 mutants suppress gain-of-function PPK-1 (PI4P5K) phenotypes, most likely through changes in the localisation of the ion channels. Our current model involves RHO-1 binding to and activating PPK-1, increasing levels of PIP2 and hyperactivating the NCA-1/NCA-2 channels. We suspect this ion channel complex may regulate the release of neuropeptides involved in locomotive behaviour.

Epithelial tissue morphogenesis relies on tightly regulated cell shape changes and local cell-cell contact rearrangements. However, our knowledge on how these cells’ behaviours are controlled and implemented during organogenesis remains incomplete. To address this issue, I have made use of the genetically amenable developing Drosophila eye. Patterning of the Drosophila eye is initiated by the morphogenetic furrow (MF), a developmental compartment characterized by a wave of apically constricted cells that travels from the posterior pole of the disc. In the wake of the MF emerges a regular array of aligned photoreceptor-precursor cells that will further assemble into mature ommatidia, the building blocks of the fly’s compound eye. My thesis work is concerned with characterizing the cellular and molecular mechanisms directing early ommatidia patterning. My work demonstrates that the alignment of the ommatidial precursor cells is directed by the basic helix-loop-helix transcription factor Atonal (Ato). In the wake of the MF, interfaces between ato-expressing and non-expressing cells promote the planar polarization of the acto-myosin (MyoII) cortex along the anterior-posterior axis. Consequently, MyoII is required to establish the complementary polarisation of the zonula adherens factors ECadherin (E-cad) and Bazooka (Baz) and to direct multicellular alignment. Further, my work indicates that in parallel, transcription downstream of Epidermal growth factor receptor (EGFR) signalling is required to sustain MyoII planar polarization as the clusters of aligned cells evolve toward more mature ommatidia. Importantly, both Ato and EGFR modulate E-cad levels in the wake of the MF and we propose that differences in ECad expression create interfacial tensions at the boundary between the MF and more posterior cells. In my model, this provides the directionality for polarizing MyoII. I propose that this cell response directs the alignment of group of cells such that the subsequent orderly and stereotyped EGFR-dependent recruitment of photoreceptors can proceed.

To establish morphological polarity, a cell must form a rear that is distinct from its front. For subsequent directional migration it must continuously retract its rear and protrude its front, polarised in the direction of migration. Here, two methods for the analysis of the transition from a pre-polar to a polarised state are used; spontaneous and cue dependant polarisation. Time-lapse microscopy of primary chick embryo heart fibroblasts expressing GFP-actin showed that, prior to cell polarization, actin filament bundles in the cell body reorganise to form oriented filament bundles spanning the entire cell body. These actin bundles are already known to be necessary for cell migration. It was established that the first visual step in polarity acquisition was the organisation of actin from a disordered, non-oriented arrangement to an oriented array involving a range of acto-myosin II filament dynamics. This organisation was required to specify the position of the cell rear in spontaneous and in cue dependant polarity initiation. On average it took fibroblasts 5 minutes to spontaneously polarise once acto-myosin filaments were oriented. For polarization a localised net protrusion occurred at one end of the actin filament bundles, lagging retraction of the rear. Consequently, fully polarised cells started to migrate in the direction of the long axis of the acto-myosin II filament bundles. Jasplakinolide treatment and the introduction of either constitutively active LIM kinase or dominant negative ADF/Cofilin blocked formation of oriented actin filament bundles, with cells subsequently failing to polarise. It is therefore concluded that formation of oriented actin filament bundles in the cell body of fibroblasts requires ADF/Cofilin family of proteins, and is an early event needed to co-ordinate the spatial location of the cell rear and front during fibroblast polarization.

Neurofibromatosis type 1 (NF1) is a common genetic disorder that predisposes to the development of heterogeneous tumours of Schwann cell origin, termed neurofibromas. Neurofibromas are thought to arise from a combination of genetic events – loss of the Ras-GAP neurofibromin in the Schwann cell lineage – and microenvironmental cues. Schwann cells are specialised cells that ensheath and myelinate the axons in the peripheral nervous system (PNS). In the adult they are present in a quiescent state, however following damage to the PNS they have a remarkable ability to regenerate. Distal to the site of injury, Schwann cells dedifferentiate to a progenitor-like state, in which they contribute to nerve repair by recruiting a robust inflammatory response and helping axons return to their targets. Work from our laboratory has shown that activation of the Ras/Raf/ERK pathway plays a central role in driving the switch in Schwann cell state from a fully differentiated to a proliferating, “progenitor-like” cell. Crucially, neurofibromas resemble injured nerves in that they are composed of a mixture of inflammatory cells and Schwann cells that are found dedifferentiated and dissociated from axons, suggesting that deregulation of Ras/ERK may trigger tumourigenic events. In this thesis I present work on how the Ras/Raf/ERK pathway may be regulated in Schwann cells. I show that the phosphatase MKP3 may be involved in controlling the levels of ERK activity in Schwann cells during differentiation and following nerve injury. I also describe a new model for neurofibroma formation. Using transgenic mice I show that Nf1 loss in adult, myelinating Schwann cells has no effect on peripheral nerves and does not induce tumourigenesis. However, when coupled with an injury, the mice developed tumours at a high frequency. Furthermore, I show that in the absence of Nf1, ERK signalling is deregulated upon injury, implicating this pathway in the tumour formation. This may have therapeutic relevance, which is currently being tested in our animal model. In addition, we observed that tumours only arise at the wound site, despite Schwann cells dedifferentiating along the length of the nerve. This strongly implies that the microenvironment is a crucial player in the outcome of Nf1 loss and reveals this new animal model as a promising system to further dissect molecular events involved in tumourigenesis.

The developmental refinement of an epithelium requires finely balanced rates of growth and cell loss. However, the molecular mechanisms that regulate the achievement of homeostasis, which are likely to be deregulated in tumorigenesis, remain poorly understood. In this work using the fly notum as a model system and laser cutting experiments to test in vivo tissue mechanics, I describe a novel process of live cell delamination that counter-balances tissue growth to ensure the achievement of mechanical equilibrium in the final phases of development. The fly notum is an ideal system to study this type of mechanical buffering since it remains approximately constant in size during the final phases of tissue refinement in development, whilst requiring cell growth, division and cell loss. Individual cells leaving overcrowded regions of the notum by live cell delamination follow a path of progressive junctional and apical area loss, in a 2-step mechanism that is independent of cell death. Cells first undergo serial junctional loss, leading to a cell with a small apex and few sides, followed by Myosin-II driven apical extrusion. This process of live cell delamination can be recapitulated by a simple vertex model of epithelial mechanics, where pressure is relieved as cells leave the tissue via a series of stochastic neighbor exchange events. These findings suggest that crowding-induced live cell delamination is a generic mechanism that buffers epithelia against variations in growth. This has important implications for our understanding of homeostasis and its deregulation in cancer, as well as for cancer cell invasion and metastasis.

Cancer is a disease caused by transformation of cells by the activation or over-expression of oncogenes such as Ras and c-myc, and the loss of tumour suppressor genes such as E-cadherin and scribble. The initial stage of tumourigenesis is the transformation of a single cell in an otherwise normal epithelium. What occurs at this stage is largely unknown - do the transformed cells and normal cells co-exist or is there an antagonism between them? This thesis examines the fate of epithelial cells that lose the tumour suppressor scribble when in an otherwise normal epithelium. The fate of scribble knockout clones has been studied in Drosophila melanogaster larval imaginal discs. It has been observed that scribble knockout clones are removed from the larval tissues by c-Jun N-terminal kinase (JNK) dependent apoptosis. It is though that this is an innate tumour suppressive mechanism. It is therefore of great interest and importance to understand if a similar phenomenon can be seen in mammalian cells. Scribble knockdown Madin-Darby canine kidney (MDCK) epithelial cells die only when surrounded by normal MDCK cells. Dead scribble short-hairpin RNA (shRNA) cells are apically extruded from the epithelium after cell death and exhibit classical apoptotic markers such as cytoplasmic condensation, caspase 3 activation and DNA fragmentation. Extrusion of dead scribble knockout cells occurs after initiation of apoptosis as blocking myosin activation results in many dead scribble knockout cells staying in the epithelial monolayer. Prior to cell death they maintain normal cell-cell adhesion with their normal MDCK neighbours and activate the stress induced protein kinase p38, but not c-­‐Jun N­‐- terminal kinase (JNK).

Schwann cells are found in close proximity with axons from an early developmental stage, where, in adult nerve, they exist as either myelinating or non-myelinating Schwann cells. Reciprocal, contact-dependent signalling, between Schwann cells and axons, is central to the regulation of Schwann cell proliferation, survival and differentiation, as well as axonal survival. Cell adhesion molecules (CAMs) mediate homotypic and heterotypic interactions. They are required during development, in homeostatic nerve and in nerve repair following injury. Dysregulation of signal pathways and resulting aberrant CAM expression, can lead to irreversible Schwann cell/axonal dissociation, which is a hallmark of various peripheral neuropathies and nerve sheath tumours, e.g. neurofibromas in NF1 patients. In this thesis, I conducted a microarray screen to identify early mediators of Schwann cell/axonal interaction, using a Large-T (LT)-expressing Schwann cell that had spontaneously lost the ability to interact with axons, termed LT-derived (LTD) cells. This analysis revealed that multiple cell adhesion genes had become dysregulated including N-cadherin, Semaphorin-4F, Necl-4, NCAM and L1-CAM. This shift in adhesion profile suggested that a transcription factor, for example Sox2, might be the genetic lesion responsible; however, Sox2 was found not to be responsible for the LTD phenotype, although over-expression of Sox2 altered N-cadherin localisation at Schwann cell-cell junctions. Further study showed that N-cadherin was required for homotypic interactions and was an important mediator of heterotypic interactions, where heterologous N-cadherin expression in fibroblasts was sufficient to induce fibroblasts to recognise and partially associate with axons. In addition, N-cadherin was implicated in the regulation of the cell cycle; while N-cadherin silencing, in Schwann cells prior to axonal contact, was found to impede myelination in vitro. Finally, this work showed that N-cadherin and Semaphorin-4F operate at distinct stages of the interaction process, with N-cadherin mediating axonal recognition and Semaphorin-4F involved in stabilising the Schwann cell/axonal association.

Mitochondria have their own protein synthesis machinery that synthesises the oxidative phosphorylation components encoded by their mtDNA. This translation process consists of four main phases: initiation, elongation, termination and ribosome recycling. Termination and its control have been the least investigated. Recently, however, the termination factor, mtRF1a, has been characterised as sufficient to release all the nascent proteins from the mitoribosome. Furthermore, bioinformatics has identified three additional members of this mitochondrial release factor family namely, mtRF1, C12orf65 and ICT1. The latter is now known to be incorporated into the mitoribosome but its exact function remains unclear. My project has therefore focussed on characterising the remaining two factors; mtRF1 and C12orf65, and investigating their possible involvement in mitochondrial protein synthesis. It has been demonstrated that protein synthesis is not perfect and bacterial ribosomes not infrequently stall during translation. This can result from limiting amounts of charged tRNAs, stable secondary structures, or truncated/degraded transcripts. Ribosome stalling has been shown to cause growth arrest. In order to prevent that and maintain high efficiency of mitochondrial protein synthesis such stalled complexes need to be rapidly recycled. Bacteria have developed at least three distinct mechanisms by which ribosomes can be rescued. Contrastingly, despite the presence of truncated mRNAs in mitochondria, no such quality control mechanisms have been identified in these organelles. This study investigates the potential role of mtRF1 and C12orf65 in quality control of protein synthesis in mitochondria. Both mtRF1 and C12orf65 demonstrate conservative motifs which would suggest their potential role in ribosome rescue. My findings indicate that the conserved motifs in mtRF1 are crucial to maintain normal cell metabolism and that its mutated forms negatively affect cell growth. Since these motifs are required for ribosome dependent peptidyl-tRNA hydrolysis, the data presented strongly imply that mtRF1 plays a crucial role in intra-organellar protein synthesis.

No description available.

Mechanical signals affect and regulate many aspects of the cell behaviour, including growth, differentiation, gene expression and cell death. This thesis investigates the manner by which mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, a combined fluorescence-atomic force microscope (AFM) was used to produce well defined nanomechanical perturbations while simultaneously tracking the real-time motion of fluorescently labelled intracellular organelles in live cells. By tracking instantaneous displacements of mitochondria far from the point of indentation, insights can be gained into the long-distance propagation of forces and the role of the cytoskeleton in force transmission. Quantitative analysis and tracking of mitochondria, using several image registration and tracking techniques, revealed an increase of approximately 40% in the mean mitochondrial displacement following AFM perturbation. Furthermore, when either the actin cytoskeleton or microtubules were disrupted using anti-cytoskeletal drugs, no significant change in mitochondrial displacement was observed following indentation, revealing the crucial role of both cytoskeletal networks in the long-distance transmission of forces through the cell. In addition, the effect of retinol and conjugated linoleic acid (CLA), compounds that have diverse effects on various cellular processes, on the mechanical behaviour of the cell was examined: both compounds were found to have a significant detrimental effect on the formation of focal adhesions, which was directly correlated to the measured cell elasticity (Young’s modulus) of the cell. Furthermore, quantification of mitochondrial displacements in response to applied AFM perturbations showed force propagation through the cytoskeleton to be blunted. Treatment of the two compounds in combination showed an additive effect. These results may broaden our understanding of the interplay between cell mechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease.