Insulin stimulates glucose uptake in muscle and adipose tissue by translocating glucose transporter 4 (GLUT4) from an intracellular storage sige to the cellsurface. Studies have shown that insulin-activation of PI3K is required for stimulatin of GLUT4 translocation, however, platelet-derived growth factor (PDGF) also activates PI3K in adipocytes without significant effects on GLUT4 localisation. The aim of this study is to examine the hypothesis that insulin stimulates anunique signalling pathway that regulates GLUT4 translocation in adipocytes, and to identify novel signalling candidates in this pathway. A proteomic approach was employed to compare the effect of insulin versus PDGF on protein phosphorylation in 3T3-L1 adipocytes, using high-resolution two-dimensional gel electrophoresis (2-DE) in combination with 32P-metabolic labelling and autoradiography. The effect of wortmannin, an inhibitor of PI3K, on insulin-induced phosphorylation was also examined as a further screen for selection of candidate phosphoproteins for insulin-stimulation of GLUT4 translocation. Three subcellular Insulin stimulates glucose uptake in muscle and adipose tissue by translocating glucose transporter 4 (GLUT4) from an intracellular storage site to the cell surface. Studies have shown that insulin-activation of PI3K is required for stimulation of GLUT4 translocation, however, platelet-derived growth factor (PDGF) also activates PI3K in adipocytes without significant effects on GLUT4 localisation. The aim of this study is to examine the hypothesis that insulin stimulates an unique signalling pathway that regulates GLUT4 translocation in adipocytes, and to identify novel signalling candidates in this pathway. fractions were extensively examined, the plasma membrane (PM) fraction, the cytosol fraction and a high speed pellet (HSP) fraction that contains the insulin-responsive pool of GLUT4. These studies showed that phosphorylation of 18 proteins were consistently increased by insulin treatment, with 8 proteins in the cytosol, 10 proteins in the HSP and none in the PM. The phosphorylation of 6 cytosolic and 2 HSP insulin-stimulated phosphoproteins were also significantly increased by PDGF treatment. Wortmannin inhibited the insulin-stimulated phosphorylation of all but 2 proteins. Ten insulin-stimulated phosphoproteins were wortmannin-sensitive and PDGF-insensitive, and were selected as potential candidates for involvement in insulin-stimulated GLUT4 translocation. The molecular identity of insulin-stimulated phosphoproteins were initially examined by immunoblotting. Two cytosolic 2D gel spots (C65 and C79) were identified as mitogen-activated protein kinase (MAPK) isoforms by immunoblotting. Importantly, treatment-induced changes in the phosphorylation of these spots matched those reported for MAPK in adipocytes, confirming that our phosphoprotein mapping accurately reflects cellular phosphorylation. As the observed molecular mass and isoelectric point (pI) of most insulin-regulated phosphoproteins did not match known insulin-signalling proteins, we attempted to identify these by protein sequencing. In agreement with the low abundance of signalling proteins, most of the insulin-regulated phosphoprotein spots were not Coomassie-stained, even on preparative 2D gels. Thus, corresponding spots were pooled from numerous preparative 2D gels, before tryptic digestion and anlaysis by liquid chromatography-coupled tandem mass spectrometry (LC-MS). One of the more abundant candidate phosphoproteins was identified as ATP-citrate lyase (ACL), an enzyme involved in fatty acid synthesis previously shown to be an insulin-regulated phosphoprotein. Insulin treatment also results in the dephosphorylation of several phosphoproteins, one of which was identified as eukaryotic translation elongation factor 2 (eEF2). ACL and eEF2 phosphorylation were found to be regulated in an insulin-specific and wortmannin-sensitive manner in adipocytes. Recent studies suggest that protein kinase B (PKB) is activated downstream of PI3K, and participates in insulin-stimulated glucose uptake. However, the molecular mass and pI of phosphoproteins mapped in this study did not match any of the known PKB isoforms. To investigate the regulation of PKB isoform in adipocytes, studies were performed with PKB antibodies. PKBb expression was found to be induced upon differentiation of 3T3-L1 adipocytes, where insulin, but not PDGF stimulated its phosphorylation. In contrast, PKBa expression was high in fibroblasts, where PDGF was more efficacious than insulin in inducing its phosphorylation. These results suggest that PKBb, rather than PKBa, is the most pertinent isoform for insulin action in adipocytes. PDGF exerted no detectable effect on the phosphorylation of PKBb in 3T3-L1 adipocytes, as determined by phospho-specific antibodies, 32P-labelling and shifts in electrophoretic mobility and pI. Furthermore, insulin stimulated the translocation of PKBb to membrane fractions of adipocytes, whereas PDGF was without effect. Together, these results strongly suggest that PDGF does not activate PKBb in 3T3-L1 adipocytes. Using 2-DE, we have obtained data which support the hypothesis that insulin stimulates an unique signalling pathway in adipocytes. Three potential models are proposed to explain the insulin specificity: targeting of PI3K to pre-assembled signalling complexes in the HSP, activation of specific PI3K isoforms, and activation of two PI3K-dependent signalling pathways. These models remain to be validated in future studies. Candidate phosphoproteins for insulin regulation of glucose metabolism have been mapped using 2-DE. While only abundant metabolic enzymes have been identified so far, methods for phosphoprotein purification and identification have been established. The candidate phosphoproteins mapped in the HSP fraction are likely to be low abundance signalling proteins, and further preparative isolation of these phosphoproteins is required in order to obtain enough protein for identification of these candidates. In conclusion, the present study has demonstrated the successful application of proteomic techniques in cell signalling research.

Urine contains exosomes originating from the circulation and all cells lining the urinary tract. Exosomes are a route of inter-cellular communication along the nephron potentially able to transfer of protein and/or RNA. It is not known whether this is a regulated process analogous to other cell-to-cell signalling systems. The aims of this study were to develop nanoparticle tracking analysis (NTA) as a technique to quantify exosomes in urine. Secondly, the hormonal regulation of exosome uptake in vitro and in vivo was investigated. Thirdly, exosome excretion in a central diabetes insipidus (DI) patient and a patient group after radiocontrast exposure was measured to investigate exosome excretion along the kidney in injury. Using the fluorescent capabilities of NTA, urinary exosomes were quantified in urine samples. NTA was able to detect changes in aquaporin 2 levels in vitro and in vivo. Storage conditions for human urinary exosomes were also optimised using NTA. A kidney cortical collecting duct cell line (CCDs) was used to model regulation of exosome uptake in vitro. CCDs were stimulated with desmopressin, a vasopressin analogue, and uptake of fluorescently-loaded or microRNA-loaded exosomes was measured. Desmopressin stimulated exosome uptake into collecting duct cells via V2 receptor stimulation. Intra-cellular uptake of exosomes was confirmed by microRNA specific mRNA down-regulation. Mechanistically, exosome uptake in response to desmopressin required cyclic AMP production, was mediated by clathrin-dependent endocytosis and was selective for exosomes from kidney tubular cells. In mice, fluorescently-loaded exosomes were systemically injected before and after administration of the V2 antagonist, tolvaptan, and urinary exosome excretion was measured. Basally, 2.5% of injected exosomes were recovered in urine; tolvaptan treatment resulted in a 5-fold increase. By combining antibodies to nephron segment-specific proteins with NTA we measured human urinary exosome excretion in central diabetes insipidus (DI) and after radiocontrast exposure (n=37). In DI, desmopressin reduced the excretion of exosomes derived from upstream glomerular and proximal tubule cells. In patients exposed to radiocontrast, urinary exosomes from the glomerulus were positively correlated with the tubular injury markers KIM- 1 and NGAL. These findings therefore show that tubular exosome uptake is a specific, hormonally regulated process that is reduced with injury. Physiologically, exosomes are a mechanism of inter-cellular communication; therapeutically, exosomes represent a novel vehicle by which RNA therapy could be targeted for the treatment of kidney disease.

Conventional or “primed” human embryonic stem cells (hESCs) rely on FGF and TGFβ signalling for self-renewal, and occupy a developmentally advanced state of pluripotency comparable to mouse EpiSCs. Recent reports demonstrate that a naïve state of human pluripotency can be consistently derived either through transient histone deacetylase inhibition mediated resetting of conventional hESCs or via isolation of the inner cell mass. Long-term propagation of this state can be achieved using a cocktail of MEK, GSK3 and PKC inhibition in conjunction with leukaemia inhibitory factor (LIF) supplementation (t2iLGö) and a feeder layer of inactivated mouse embryonic fibroblasts. However, the way in which this signalling environment is interpreted in order to maintain naïve pluripotency remains unclear. I demonstrate a substrate consisting of a high concentration of tissue-derived laminin in combination with t2iLGö is sufficient to replace the feeder layer. Cultures maintained under these conditions are karyotypically normal, maintain a naive pluripotent transcriptional profile and exhibit reduced aberrant expression of mesodermal and endodermal lineage markers. I utilise the increased stringency of this culture system in combination with small molecule inhibitors to examine the roles of FGF, Activin/Nodal and JAK/STAT signalling in human naïve pluripotency. Naïve hESCs proliferate and maintain pluripotency marker expression in the presence of FGF receptor inhibition. In contrast, TGFβ signalling inhibition leads to rapid downregulation of human specific naïve pluripotency marker, KLF17, followed by the eventual collapse of the naïve transcription factor circuitry. Naïve hESCs self-renew in both the absence of LIF and presence of JAK/STAT inhibitors. However, further investigation of JAK/STAT signalling identified the increased potency of Interleukin 6 (IL-6) over LIF to activate the JAK/STAT pathway. Supplemental IL-6 improves colony-forming capacity under self-renewing conditions and attenuates differentiation following inhibitor withdrawal. Furthermore, prolonged activation of IL-6 signalling suppresses expression of GATA2 and GATA3 and upregulates KLF4 transcripts. Finally, I investigate whether ablation of PKCι is sufficient to replace the activity of the PKC inhibitor, Gö6983. Established naïve cultures that are PKCι null continue to express naïve markers and suppress upregulation of lineage makers following withdrawal of Gö6983. Furthermore, ablation of PKCι in conventional ESCs enables the maintenance of NANOG expression and the emergence of KLF17 expression in the absence of Gö6983 during histone deactylase mediated resetting.

Ca2+ signals determine smooth muscle contraction and the switch from a contractile to a migratory-proliferative phenotype(s), which requires changes in gene expression. However, the mechanism by which different Ca2+ signals are selective for these processes is enigmatic. In the thesis, I built on the “panjunctional sarcoplasmic reticulum” hypothesis, and described the evidence in support of the view that a variety of Ca2+ pumps and release channels, with different kinetics and affinities for Ca2+, are strategically positioned within the cytoplasmic nanocourses of pulmonary arterial smooth muscle cells (PASMCs), and they serve to demarcate different Ca2+ signalling. Nanocourses of the SR are formed in the perinuclear, extraperinuclear, subplasmalemmal regions and the nucleus. Different subtypes of ryanodine receptors (RyRs) are targeted to those nanocourses. Immunocytochemistry results suggest that RyR1s was preferentially targeted to the subplasmalemmal and nuclear nanocourses of PASMCs, they gave rise to a spatially restricted Ca2+ signal within the nanocourses upon stimulation, without affecting global Ca2+ concentration. The Ca2+ signals in the subplasmalemmal nanocourses were shown to induce arterial smooth muscle cell relaxation. On the other hand, the RyR2 and 3 were shown to target to the perinuclear and extraperinuclear nanocourses. Upon stimulation, they generate propagating Ca2+ waves in the cytoplasmic nanocourses, which trigger arterial smooth muscle cell contraction. However, during this process, no Ca2+ transient was observed within the subplasmalemmal nanocourses, suggesting that the regulation of both contraction and relaxation of smooth muscle cells are achieved by spatially restricted Ca2+ signals within different nanocourses. Invaginations of the nucleoplasmic reticulum in arterial myocytes form trans-nuclear networks of cytoplasmic nanospaces, generate Ca2+ signals by strategically positioned Ca2+ pumps (SERCA1) and release channels (RyR1). Within a subpopulation of nuclear invaginations, evoked Ca2+ signals via ryanodine receptors exhibited spatial and temporal separation from adjacent Ca2+ signals within a single “activated” nuclear invagination, and also from those Ca2+ signals arising within different nuclear invaginations. Moreover, nuclear invaginations provide sites for transcriptional suppression, because lamin A and/or emerin line the entire surface of their inner nuclear membranes and co-localise with nesprin-1 positive puncta. More intriguing still, a subpopulation of these nuclear invaginations harboured punctate regions of colocalisation between lamin A and the suppressive heterochromatin mark H3K9me2, while emerin-positive invaginations harboured puncta of BAF (Barrior to autointegration factor) co-localisation and thus an alternative pathway to the regulation of gene expression. I propose that nuclear invaginations form cytoplasmic nanotubes within which nano-patterning of Ca2+ signals may support stochastic modulation of transcriptional suppressors. Together, the cytoplasmic nanocourses form a cell-wide web for Ca2+ signalling and the regulation of various arterial smooth muscle functions, ranging from the regulation of blood pressure by vasodilation and vasoconstriction to gene expression.

CD43, a representative of the leukocyte mucin family proteins, is a transmembrane protein highly expressed on most lymphohemopoietic cells and is believed to play a role in the regulation of leukocyte activation and/or migration. CD43 was shown to be proteolytically shed from human cells and high concentrations of soluble CD43 have been found in human plasma. The biological significance of CD43 shedding however remains enigmatic. To study the functional significance of CD43 shedding, we initiated our study by investigating whether CD43 shedding also occurs in the murine system and confirmed using flow cytometry, Western blot and ELISA techniques that murine CD43 is cleaved from the cell surface as is observed in the human system. To examine the biological significance of CD43 shedding, we designed and constructed non-sheddable forms of murine CD43. Ectopic expression of non-sheddable CD43 molecules in primary CD43 deficient bone marrow cells showed that these CD43 mutants have serious negative impacts on cell viability, revealing CD43 shedding as an essential process and implying that the CD43 mutants interfered with intracellular signaling processes. Our data support the hypothesis that CD43 ectodomain shedding is a requirement for release of the cytoplasmic domain and its translocation to the nucleus. In support of our hypothesis, we confirmed that the CD43 cytoplasmic domain is localized in the nucleus and is modified by SUMO (small ubiquitin-like modifier) peptides. In an attempt to determine the functional significance of CD43 nuclear translocation and SUMO modification, we examined nuclei from hemopoietic cells more closely and observed that the CD43 cytoplasmic tail is localized in a subnuclear structure called promyelocytic nuclear bodies, which control many nuclear functions including apoptosis. Consistent with this observation we find that leukocytes from CD43 deficient mice have an increased apoptotic response upon growth factor withdrawal. We conclude that nuclear translocation of the CD43 cytoplasmic tail serves to control the apoptotic response in leukocytes and that CD43 functions as an anti-apoptotic molecule. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Atrial fibrillation (AF), the most common sustained arrhythmia, is associated with abnormal intracellular Ca2+ handling. Understanding AF requires comprehensive understanding of ionic currents, Ca2+ handling, phosphorylation regulation and related signalling pathways, but appropriate models are limited. The aim of this thesis is to develop an ionic model of the mouse atrial myocyte to investigate the cellular proarrhythmic mechanisms. We have developed the first mouse atrial myocyte model that incorporates mathematically detailed ion channels, cellular Ca2+ and Na+ handling and their regulation by Ca2+-calmodulin-dependent protein kinase II (CaMKII) and protein kinase A. For the first time, the inositol 1,4,5-trisphosphate (IP3) production system and its effects on excitation-contraction coupling have also been described. The validated model predicted that: 1) hyperactivity of CaMKII and elevated intracellular Na+ concentration are the crucial factors that induce sarcoplasmic reticulum (SR) Ca2+ spontaneous release and delayed afterdepolarisations; 2) β-adrenergic stimulation may have proarrhythmic effects by exacerbating Ca2+ overload; and 3) enhanced activity in ryanodine receptors during IP3-induced Ca2+ release is the major cause of the arrhythmogenesis in IP3 signalling.

Neutrophils form a first line of defence against infections. These short-lived, terminally differentiated cells perform many important functions, including chemotaxis, degranulation, reactive oxygen species (ROS) release and cytokine production. Whilst neutrophils are essential for host immunity, their inappropriate recruitment, activation and/or removal can contribute to excessive inflammation and host damage, as exemplified in autoimmune diseases such as rheumatoid arthritis. It is therefore essential that neutrophil function is tightly regulated. Neutrophils are activated by a range of stimuli, including immune complexes. Neutrophil functions are tightly regulated by intracellular signalling events that are induced by the ligation of cell surface receptors, for example, the binding of immune complexes to Fc receptors. Phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk) are key signalling intermediates that act downstream of many cell surface receptors. They are involved in the regulation of numerous biological processes in the neutrophil. Using pharmacological interventions, I analysed PI3K signalling in immune complex-stimulated human neutrophils and uncovered a previously uncharacterised, noncanonical signalling pathway, PI3K-Cdc42-Pak-Mek-Erk. This represents an unusual situation where Pak acts as the MAP3K downstream of Cdc42 in a PI3K-dependent fashion. By performing a range of functional experiments, I showed that this unconventional signalling pathway promotes apoptosis in human neutrophils by regulating the ratio between anti- and pro-apoptotic members of the Bcl-2 family proteins. No other immune complex-induced, PI3K-dependent neutrophil function tested depended on PI3K-Cdc42-Pak-Mek-Erk signalling. Mouse knock-outs of all components of this signalling pathway have been described. Immune complex-induced apoptosis was also PI3K-dependent in mouse neutrophils, but experiments performed with inhibitors showed that, in contrast to human neutrophils, this was not dependent on PI3K-Cdc42-Pak-Mek-Erk signalling. The myeloid leukaemia cell line, PLB-985 is amenable to knock-down and can be differentiated to become neutrophil-like. These cells are not notably activated by immune complexes, perhaps because they do not express the major Fcγ receptor, CD16. Since retroviral expression of CD16 in PLB985 cells did not improve their response to activation by immune complexes, I was not able to confirm my observations with human neutrophils genetically. Collectively, I showed that a novel, pro-apoptotic signalling pathway operates downstream of Fcγ receptors in the human neutrophil. The fact that this signalling pathway appears to regulate apoptosis specifically suggests uncoupling pro- and anti-inflammatory effects induced by immune complexes might be possible. This may be helpful in the design of improved therapies of autoimmune diseases such as rheumatoid arthritis, in which immune complex-driven neutrophilic inflammation contributes to disease pathogenesis and where neutrophil apoptosis is disturbed.

Intercellular signalling is essential for multicellular organisms to coordinate growth and development, and is mediated by a huge variety of proteins. Some signalling pathways rely on the proteolytic cleavage of membrane proteins by a relatively newly discovered process of regulated intramembrane proteolysis (RIP), the cleavage of proteins within a transmembrane domain. There are four classes of intramembrane cleaving proteases (ICliPs) – Rhomboids, Site-2-proteases, Signal peptide peptidases and γ-secretase. Of all the ICliPs studied to date, γ-secretase is unique, as it is comprised of a four-protein complex, and is only found in multicellular organisms. A vast amount of research is carried out on the γ-secretase complex, not just because of its role in developmentally important pathways, such as NOTCH signalling, but also due to its role in Alzheimer’s disease. The β-amyloid precursor protein (APP) is cleaved by γ-secretase, and defects in this process result in the release of abnormal peptides that form the senile plaques in the brains of Alzheimer’s disease patients. Homologues of the four components of γ-secretase (PRESENILIN (PS), NICASTRIN (NCT), ANTERIOR PHARYNX DEFECTIVE-1 (APH-1) and PRESENILIN ENHANCER-2 (PEN-2)) are found in plants. The aim of this thesis was to characterise the potential γ-secretase components in Arabidopsis thaliana, to determine whether they form a complex, and to analyse what role, if any, they play in plant signalling. The members of the putative Arabidopsis γ-secretase complex (AtPS1 and 2, AtNCT, AtAPH1 and AtPEN2) were identified through BLAST searches, and found to be uniformly expressed. Analysis of T-DNA insertion mutants in each of these genes, and combinations there of, revealed no gross morphological differences to wild type under normal growth conditions and when subjected to a range of stresses. Protein fusions to GFP under the control of the 35S promoter were constructed and stably transformed into plants. AtPEN2:GFP is expressed throughout the plant, and accumulates in BFA sensitive Golgi bodies in roots. AtPS1:GFP, only accumulates strongly in developing seeds. Native blue PAGE was used to look for high molecular weight complexes (HMW) containing AtPEN2:GFP and AtPS1:GFP. Both fusion proteins were found in similar sized HMW complexes. A variety of methods were used to look for substrates of the iv putative γ-secretase complex in Arabidopsis, and although no specific substrates were identified, a potential role in seed development has been established.

Wnt signalling is a critical cellular communication pathway controlling cell fate in all metazoan organisms. Timely activation of this pathway is crucial to coordinate development, control homeostasis of adult tissues, and to avoid cancer. Wnt signal transduction depends primarily on the activities of three multiprotein complexes; the 'degradasome', which targets the central effector β-catenin for degradation in the absence of Wnt; the 'signalosome', which is assembled by Dishevelled upon Wnt-receptor binding to inactivate the degradasome, thus allowing β-catenin to accumulate; and the 'enhanceosome', which captures β-catenin, granting it access to target genes and relieving their transcriptional repression by Gro/TLE. Many of the components of these complexes have now been identified, but details of their regulation, and in particular the mechanisms by which they are switched on and off, remain poorly understood. The majority of this thesis is concerned with the mechanism by which β-catenin relieves the transcriptional repression imposed upon Wnt target genes, and thereby activates the Wnt 'transcriptional switch'. In Chapter 2, I present data showing that apposition of Gro/TLE and UBR5, a HECT E3 ubiquitin ligase, by β-catenin promotes Gro/TLE ubiquitylation, earmarking it for extraction by the VCP/p97 ATPase and ultimately leading to inactivation of its repressive function. In Chapter 3, I present the results of a different, ongoing study to identify the mechanism by which a cytoplasmic negative regulator, Naked, acts to interfere with the function of Dishevelled, promoting the switching of signalosomes and the termination of canonical Wnt signalling. These findings advance our understanding of the mechanisms by which the Wnt signalling pathway is switched on and off, and suggest new targets for therapeutic intervention in Wnt- driven cancers.

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