Master of Science / Department of Physics / Robert Szoszkiewicz / For proteins in living cells, forces are present at all levels. These range from macroscopic to single molecule levels. Single molecule atomic force microscopy (AFM) in force extension (FX) and force clamp (FC) modes can investigate the mechanical properties of proteins, for example, forces at which proteins unfold, or the kinetics of these processes. In the FX-AFM experiments, proteins are pulled at constant velocity, while in FC-AFM experiments, proteins are pulled at constant force. This thesis describes i) how a single molecule FX/FC-AFM was constructed using various components, ii) how it was calibrated and tested using (I27)4 polyprotein, and iii) how it was applied to the studies of a Notch construct. Building up the single molecule FX/FC-AFM system opened a path to investigate the mechanical properties of proteins. Such a system was tested on a known protein construct, hence the usage of the (I27)4 polyprotein. The Notch protein is a signaling protein that plays a role in triggering breast cancer. It is believed that understanding the mechanical properties of Notch can help to understand its oncogenic functions. We have successfully constructed and calibrated the FX/FC-AFM setup. It was found that the AFM worked for the standard calibration protein of (I27)4. The results on a Notch construct revealed our ability to see some conformational transition state in this molecule under force. These results opened a path for further investigations of a Notch construct at various physiologically relevant conditions.

In contrast to mammals, adult zebrafish display cellular regeneration of lost motor neurons and achieve functional recovery following a complete spinal cord transection. Using adult zebrafish as a model to study how key developmental pathways can be re-activated to regulate neuroregeneration in cellular recovery, I addressed the following questions: 1) What is the role of Notch signalling during regenerative mechanisms in the lesioned spinal cord of the adult zebrafish? 2) What is the role of Notch overexpression in neurogenesis in the adult zebrafish retina? 3) Which additional signalling pathways are involved in the generation of motor neurons during spinal cord regeneration in adult zebrafish? 1) In the main part of my thesis I have investigated the role of Notch signalling during spinal cord regeneration. The Notch pathway has been shown to regulate neural progenitor maintenance and inhibit neuronal differentiation in the vertebrate nervous system. In the injured mammalian spinal cord, increased Notch signalling is held partly responsible for the low regenerative potential of endogenous progenitors to generate new neurons. However, this is difficult to test in an essentially non-regenerating system. We show that in adult zebrafish, which exhibit lesion-induced neurogenesis, e.g. of motor neurons from endogenous spinal progenitor cells, the Notch pathway is also reactivated. I over-activated the Notch pathway by forced expression of a heat-shock inducible active domain of notch in spinal progenitor cells. I observed that although apparently compatible with functional regeneration in zebrafish, forced activity of the pathway significantly decreased progenitor proliferation and motor neuron generation. Conversely, pharmacological inhibition of the pathway increased proliferation and motor neuron numbers. Thus in summary our work demonstrates that Notch is a negative signal for regenerative neurogenesis in the spinal cord. Importantly, we show for the first time that spinal motor neuron regeneration can be augmented in an adult vertebrate by inhibiting Notch signalling. 2) While in the lesioned spinal cord, over-activation of Notch attenuated neurogenesis, I observed that in the unlesioned retina the same manipulation led to strong proliferation of cells in the inner nuclear layer, presumable Müller glia cells which are the retinal progenitor cells. This coincided with an increase in eye size in adult zebrafish. These preliminary findings provide the first hint that the role of Notch may differ for different adult progenitor cell pools and will lead to future investigations of Notch induced neurogenesis in the retina. 3) We have evidence from previous studies that the dopamine and retinoic acid (RA) signalling pathways may be involved in the generation of motor neurons in the adult lesioned spinal cord. Using in situ hybridisation, I assessed the gene expression patterns a) for all D2-like receptors and b) candidate genes that relate to the RA pathway in the adult lesioned spinal cord to identify the signalling components. a) I found that only the D4a receptor was upregulated in spinal progenitor cells in the ventricular zone rostral to the lesion site, but not caudal to it. This correlates with other results showing that dopamine agonists increase motor neuron regeneration rostral, but not caudal to a spinal lesion site. b) I observed a strong increase in the expression of Cyp26a, a RA catabolising enzyme, in the ventricular progenitor zone caudal to the lesion site, in contrast to the weak expression rostrally. Crabp2a, a cellular retinoic acid binding protein, was also upregulated rostral and in close proximity to the lesion site in a subpopulation of neurons located ventrolaterally in the spinal cord. In summary, we show that the Notch pathway negatively regulates neurogenesis in the spinal cord in contrast to the retina and provide evidence that dopamine from the brain signals via the D4a receptor to promote the generation of motor neurons in addition to RA, which may also play a role in this process. These insights into adult neural progenitor cell activation in zebrafish may ultimately inform therapeutic strategies for spinal cord injury and neurodegenerative diseases such as motor neuron disease.

Epithelial to Mesenchymal Transition (EMT) is a cellular transformation process that is employed repeatedly and ubiquitously during vertebrate morphogenesis to build complex tissues and organs. Cellular transformations that occur during cancer cell invasion are phenotypically similar to developmental EMT, and involve the same molecular signalling pathways. EMT processes are diverse, but are characterised by: a loss of cell-cell adhesion; a gain in cell-matrix adhesion; an increase in cell motility; the secretion of proteases that degrade basement membrane proteins; an increased resistance to apoptosis; a loss of polarisation; increased production of extracellular matrix components; a change from a rounded to a fibroblastic morphology; and an invasive phenotype. This thesis focuses explicitly on endocardial EMT, which is the EMT that occurs during vertebrate embryonic heart development. The embryonic heart initially forms as a tube, with myocardium externally, endocardium internally, with these tissue layers separated by a thick extracellular matrix termed the cardiac jelly. Some of the endocardial cells in specific regions of the embryonic heart tube undergo EMT and invade the cardiac jelly. This causes cellularised swellings inside the embryonic heart tube termed the endocardial cushions. The emergence of the four chambered double pump heart of mammals involves a complex remodelling that the endocardial cushions play an active role in. Even while heart remodelling is taking place, the heart tube is operating as a single-circulation pump, and the endocardial cushions are performing a valve-like function that is critical to the survival of the embryo (Nomura-Kitabayashi et al. 2009). As the endocardial cushions grow and remodel, they become the valve leaflets of the foetal heart. The endocardial cushions also contribute tissue to the septa (walls) of the heart. Their correct formation is thus essential to the development of a fully functional, fully divided, double-pump system. It has been shown that genetic mutations that cause impaired endocardial EMT lead to the development of a range of congenital heart defects (Fischer et al. 2007). An extensive review is conducted of existing experimental investigations into endocardial EMT. The information extracted from this review is used to develop a multiscale conceptual model of endocardial EMT, including the major protein signalling pathways involved, and the cellular phenotypes that they induce or inhibit. After considering the requirements for computational simulations of EMT, and reviewing the various techniques and simulation packages available for multi-cell modelling, cellular Potts modelling is selected as having the most appropriate combination of features. The open source simulation platform Compucell3D is selected for model development, due to the flexibility, range of features provided and an existing implementation of multiscale models; that include subcellular models of reaction pathways. Based on the conceptual model of endocardial EMT, abstract computational simulations of key aspects are developed, in order to investigate qualitative behaviour under different simulated conditions. The abstract simulations include a 2D multiscale model of Notch signalling lateral induction, which is the mechanism by which the embryonic heart tube is patterned into cushion and non-cushion forming regions. Additionally, a 3D simulation is used to investigate the possible role of contact-inhibited mitosis, upregulated by the VEGF protein, in maintaining an epithelial phenotype. One particular in vitro investigation of endocardial EMT (Luna-Zurita et al. 2010) is used to develop quantitative simulations. The quantitative data used for fitting the simulations consist of cell shape metrics that are derived from simple processing of the imaging results. Single cell simulations are used to investigate the relationship between cell motility and cell shape in the cellular Potts model. The findings are then implemented in multi-cell models, in order to investigate the relationship between cell-cell adhesion, cell-matrix adhesion, cell motility and cell shape during EMT.

Injury, infection and tissue malfunction are triggers of inflammation which if not regulated may acquire new characteristics that result in pathological outcomes. Since innate immunity plays a key role in the resolution of acute inflammation knowledge of the regulation of this component of the host response is relevant to understanding processes in disease progression and therefore has potential clinical benefits. In this thesis I have applied zebrafish as a model organism to investigate the response of innate immune cells to qualitatively distinct inflammatory signals in the absence of adaptive immunity. Using a zebrafish embryo wound injury model I have investigated leukocyte migration profiles by in vivo imaging. In response to wound alone leukocytes migrated to the site of injury with predominantly random walk behaviour. However, the addition of lipopolysaccharide (LPS) enhanced recruitment and influenced the directionality of leukocyte migration to the wound. I demonstrate that leukocyte dynamic behaviour is also dependent on the location of the cells. The LPS enhanced directionality and reduced the random walk behaviour of the leukocytes, and these effects were ablated in the presence of the p38 mitogenactivated protein kinase (MAPK) specific inhibitor SB203580. Cytokine gene profiling in adult zebrafish leukocytes reveals that LPS can stimulate a pro-inflammatory response via the activation of p38 MAPK characteristic of mammalian innate immune responses. It is documented in mammalian innate immune cells that LPS can modulate Notch mediated signalling and thereby cell function. Using zebrafish with null mutations in Notch, which provide an unbiased in vivo model, I have investigated the influence of Notch signalling on leukocyte recruitment and demonstrate that migration to a wound injury is reduced. However, this effect is due to decreased cell numbers and not altered function as the Notch signalling inhibitor DAPT had no effect of recruitment to wound injury. The defect in myelomonocyte numbers was also present in adult zebrafish and this was partially compensated for by an increase in lymphocytes. The experimental results that I report here highlight zebrafish as a model 2 organism for studying the function and regulation of innate immunity. The unique optical translucency, which permits in vivo imaging of host responses in real-time, facilitates the analysis of the innate immune response to different inflammatory signals and immune modulators.

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The liver is the largest solid organ in the body and is frequently the site of injury. During disease, liver injury is usually compensated for by exceptionally efficient regeneration which occurs both from differentiated epithelia and also from an undifferentiated cell population with stem cell like qualities known as hepatic progenitor cells (HPCs). HPCs are particularly active during massive or chronic liver injury and therefore are an attractive target for much needed novel therapies to enhance regeneration in patients for whom the only current effective therapy is liver transplantation. Stem cells in other organs systems are believed to reside in a specialised microenvironment or niche which supports their maintenance and function. To investigate the hypothesis that HPCs are supported by a functional niche and are capable of regenerating hepatocytes, we commenced by establishing a number of murine in vivo models. Having shown a stereotypical niche, consisting of macrophages, myofibroblasts and laminin exists in both animal models and human disease, we investigated the active recruitment of extrahepatic cells into this niche and showed that macrophages are actively recruited from the bone marrow during liver injury. Macrophages were shown to influence HPC behaviour during injury. Furthermore using macrophages as a cellular therapy, induced HPC activation with corresponding changes to liver structure and function. Investigation of signalling pathways revealed and confirmed a TWEAK dependent activation of HPCs following macrophage transfer. Having demonstrated the potential for macrophage therapy via HPC activation, we aimed to study the ability of HPCs to regenerate the hepatic parenchyma. To do so we developed and characterised a novel model of hepatocellular injury and HPC activation. Using the genetic labeling of hepatocytes in this model we were able to show rapid and large scale repopulation of hepatocytes from a precursor source with HPCs being the critical precursor source of hepatocellular regeneration. In addition this process is again dependent on TWEAK signalling, without which HPC mediated regeneration fails resulting in mortality. Therefore HPCs are an attractive biological target for regenerative medicine, and both TWEAK signalling and autologous macrophage infusion offer genuine potential to manipulate these cells as future therapies.

The Notch signalling pathway is universally conserved in all metazoan species, and is involved in many aspects of cell fate determination and tissue homeostasis, during development and in adult organisms. Several developmental diseases are associated with defective Notch signalling, and the Notch pathway has been implicated in a growing number of cancers. The Notch signalling pathway requires direct cell-cell contact for ligand binding and receptor activation to occur. Specific domains within the Notch receptors and ligands have been identified as necessary for the interaction to take place, and a series of enzymes are known to regulate Notch signalling via glycosylation. Other domains beyond the minimal ligand binding region of the Notch receptor are also known to influence binding. The aim of this study was to characterise the molecular basis for ligand binding by the Notch receptor, and how this is regulated by glycosylation. The effects on ligand binding of specific amino acid substitutions and sugar modifications were tested using prokaryotically -expressed proteins, and a series of constructs containing additional domains N-terminal of the ligand binding region was produced prokaryotically and eukaryotically to test how additional domains might affect ligand binding. Binding was assessed by a flow cytometry-based binding assay and by SPR in order to investigate how particular modifications affected ligand binding. These assays indicated that an evolutionarily-conserved hydrophobic site exists within the central β-sheet of EGF12 in the Notch receptor that is directly adjacent to the O-fucosylation site within this domain. The GlcNAc-fucose disaccharide modification at this position was found to increase binding of hNotch1 to both Jagged1 and DLL4. Additional EGF domains N-terminal to the ligand binding region showed opposite effects on binding to these two ligand classes, suggesting that the precise mode of binding may vary slightly between different Notch ligands.

The Notch signalling pathway is conserved in all metazoan species and plays a crucial role in development and tissue maintenance. Canonical Notch signalling requires cell-cell contact to allow the interaction between Drosophila Notch receptor and its ligands, Serrate and Delta. The Notch Abruptex (Ax) region comprises 24-29 of the 36 epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Mutations in the Ax region give rise to three distinct phenotypes in Drosophila. Notch EGF repeats 11-12 form the ligand binding region (LBR). The recently solved structure of the module at the N-terminal of Notch ligands (MNNL) of a human Notch ligand, Jagged1, revealed that the domain was a calcium-binding C2 domain with Ca²⁺-dependent lipid binding. This study aimed to investigate the intra- and intermolecular properties of Drosophila Notch Ax region, LBR, and the MNNL of Notch ligand Serrate. In WT Drosophila Notch EGF23-25, all three EGF domains were found to be Ca²⁺-binding, and a previously unknown Ca²⁺ binding consensus sequence was identified. Ax^N-suppressor mutations D948V and N986I were shown to impair the Ca²⁺-binding properties of the mutant EGF domain without affecting the neighbouring domains, suggesting a mechanism to explain the signalling phenotype associated with this mutation type. Notch EGF11-13 showed Ca²⁺-binding in each EGF domain and binding to ligand-expressing cells. Its C-terminal tag was found to influence the Ca²⁺-dependent fold of EGF13, suggesting a future strategy for protein expression. A Serrate fragment MNNL-EGF3 showed Ca²⁺-dependent lipid binding, which was not observed in a construct lacking MNNL. The lipid binding could be reduced by a substitution D197A in MNNL, suggesting this mutant could be used to probe functional importance of MNNL in model organism studies. Binding between Serrate and Notch was assessed with a new cell aggregation assay method using flow cytometry, and agreed with previously published studies. Binding to Delta was subsequently measured, which suggested ligand specific differences although Notch residue L504 was important for both Serrate- and Delta-binding. Collectively these studies establish that Drosophila Notch and its ligand Serrate has similar properties to mammalian homologues, which will facilitate future structural and functional studies.

The Notch receptor family plays a key role in development and disease. In cancer, Notch can act either as an oncogene or as a tumour suppressor, and possibly as a cancer stem-cell factor. Whereas most research has focused on downstream signalling events, little is known about the cell surface organisation of Notch and its ligands. The extracellular part of Notch consists mainly of 36 epidermal growth factor-like domains (EGF-domains), many of which bind calcium. Studies have shown that tandem repeats of calcium-binding EGF domains form a rigid linear arrangement; however, the lack of calcium binding in EGF6, EGF10 and EGF22 led to the hypothesis that these might be sites of flexibility. This thesis addresses the effect of these domains on the organisation of the extracellular region of Notch and provides further insight into the calcium-binding properties of Notch. NMR residual dipolar coupling (RDC) measurements of these regions are presented, together with the X-ray crystallographic data obtained in collaboration. The crystal structure of the human Notch-1 construct EGF4-7 shows a tilt angle of 90° at the EGF5-6 interface which is much larger than the tilt angles of 10-20° observed for the EGF11-13 crystal structure. RDC measurements demonstrated an angle of ~70° in solution. The crystal structures of EGF21-23 and EGF20-23 showed a rod-shaped interface for the EGF21-22 domain, in which a cis-proline forms the packing interaction to a tyrosine at the β-turn in the major β-sheet of EGF22. These two interfaces are novel and demonstrate the possibility of interface formation without Ca²⁺. Crystallisation was unsuccessful for the EGF8-11 construct. However, RDC measurements indicate interdomain motion between EGF9 and EGF10 demonstrating a flexible interface. These data establish new information on the structural organisation and calcium-binding properties of the extracellular region of Notch and identify flexible and rigid interfaces within multiple tandem repeats of EGF domains. This information will be invaluable in constructing models of Notch-ligand complexes for testing in future functional experiments.

Delta-Notch signaling is crucial for development of nearly every tissue in metazoans. Signals received by the Notch receptor influence transcription of select target genes that ultimately restrict the developmental fate of the signal receiving cell with respect to its neighbors. The Notch pathway also functions in contexts of abnormal proliferation and differentiation, e.g. cancer and inflammation. Therefore, understanding the regulation of signaling through the Notch receptor protein at the cellular and molecular level is of great significance. In this dissertation, I investigated three ways in which Notch signaling is regulated, namely (1) proteolysis of the Delta ligand; (2) endocytosis of the Delta ligand; and (3) proteolysis of the Notch receptor.. The Delta protein has three functions. First, Delta is a ligand for Notch when bound to it from an adjacent cell. Second, Delta is an inhibitor of Notch when coexpressed with it in the same cell. Third, Delta is hypothesized to be a receptor and, upon binding to Notch, signals to nucleus. Delta undergoes proteolysis by ADAM proteases and there are two contradictory models for the role of Delta cleavage: (1) cleavage disables Delta function; and (2) cleavage activates Delta function. Overall, the results presented in this dissertation strengthen the first model and weaken the second one. Consistent with the first model, we showed that preventing Delta cleavage strengthens its ligand function. As well, when co-expressed in the same with Notch, Delta cleavage is upregulated therefore disabling Delta function as inhibitor of Notch. In contrast to the second model, we showed that Delta proteolysis does not follow a previously established pattern of cleavages typical of cell surface proteins that are activated by proteolysis. Delta also undergoes endocytosis. Two general models have emerged that are again contradictory: (1) endocytosis downregulates cell surface expression of Delta and therefore diminishes its ability to bind Notch; (2) endocytosis of Delta invokes activation of Notch signaling. Overall, our results strengthen the first model and weaken the second one. In support of the first model, we first demonstrated that Notch activation shows a linear relationship to the amount of Delta ligand present on the cell surface and that subsequent inhibition of cell surface expression of Delta leads to its loss of function. In contrast to the second model, we showed that endocytosis of Delta is not required to activate Notch. We also resolved that earlier evidence in support for this model stemmed from misinterpretations of the properties of a Delta mutant protein. Proteolysis of Notch activates the signaling cascade. Binding of Delta to Notch was previously regarded as a requisite regulatory step to invoke receptor proteolysis. We identified the ability of Kuzbanian and TACE, ADAM proteases that cleave Notch in response to Delta stimulation, to activate Notch in a ligand-independent manner. Altogether, our results demonstrate that proteolysis and endocytosis of Delta are independent mechanisms that act to downregulate Delta function and are therefore an important means of attenuating the Notch signal. Alternatively, we find a novel means of enhancing Notch signals in specific contexts, namely through ligand-independent Notch activation by the ADAMs Kuzbanian and TACE. With respect to the latter observation, Kuzbanian and TACE expression is known to be elevated in several human diseases, and thus predicts that engagement of Notch signaling is a contributing factor in these pathologies.