Le manque de protéine soluble est souvent un obstacle à la caractérisation structurale des protéines. Une approche courante pour surmonter cela consiste à isoler des domaines protéiques en utilisant des méthodes itératives de création et analyse de constructions. Autrement des banques d'ADN, contenant toutes les constructions possibles d'une même protéine, peuvent être crée par troncation enzymatique et testée à la fois pour l'expression et la solubilité de celle-ci. Dans ce projet, la nouvelle méthode d'Expression des Protéines Solubles par Troncation Aléatoire Incrémentielle (ESPRIT) a été utilisée pour explorer la définition des domaines protéiques. Des protéines kinases multidomaines qui avaient échoué surexpression solubles ont été choisies pour leur intérêt biologique. La méthode a d'abord été optimisée pour améliorer la qualité et l'efficacité des banques permettant un meilleur traitement de la diversité générée. Elle a ensuite été appliquée à une protéine kinase modèle DAPK1, à partir de laquelle une définition précise de domaine a été démontrée. Le criblage des constructions a ainsi permis l'identification de protéines plus stables, et cristallisation des constructions dans les conformations alternatives. La méthode a aussi été appliquée pour identifier des variants solubles du complexe DAPK1 et son partenaire la calmoduline, permettant de mettre en évidence un domaine d'interaction minimal, plus petit que celui décrit auparavant. Alors que plusieurs tentatives pour obtenir le domaine catalytique kinase de IKKb avaient échoué, des criblages additionnels sur cette protéine avec cette méthode ont permis d'identifier des domaines de régulation solubles et fonctionnels in vivo. Enfin, il a été démontré que des constructions du domaine catalytique de p110b, faiblement exprimées dans E.coli, pouvaient être exprimées solubles dans des cellules d'insectes avec des rendements plus importants.

Biotin Protein Ligase (BPL) is an essential enzyme responsible for the covalent attachment of biotin to a specific lysine residue of biotin-dependent carboxylases, transcarboxylases and decarboxylases. Due to the fundamental processes that these enzymes are involved in such as lipogenesis, amino acid catabolism and gluconeogenesis, much research has been conducted on these enzymes. Studies encompassing structural, mutational and catalytic functions of these enzymes have lead to novel drug developments for the treatment of obesity, diabetes, metabolic syndrome, bacterial and fungal infections. As BPL is required for activation of these enzymes by biotinylation, it is believed that it too could be targeted in a similar way to produce novel therapeutics. To date, the most characterised BPLs are from the Gramnegative bacteria Escherichia coli and the archea Pyrococcus hirokoshii. However minimal information is known about other forms of clinically important bacterial species or eukaryotic forms of this important enzyme. Through my candidature I have compiled a thorough literature review summarised as chapter 1: Introduction. Furthering this literature analysis, a human BPL model was generated with aid of BPL structural co-ordinates already deposited in the protein data bank (PDB), thus allowing focus on human BPL mutations that cause multiple carboxylase deficiency (chapter 2). I have solved the structure of BPL from the clinically important pathogenic bacteria Staphylococcus aureus. This was performed in several ligand-bound and non-bound states (chapters 3 and 4). A novel high-throughput assay was developed to test BPL activity. This assay allow testing of compounds that could potentially inhibit the BPL from Candida albicans (a species responsible for invasive fungal infections) (chapter 5). Large amounts of highly purified BPL from Saccharomyces cerevisiae allowed for the first structural analysis of a eukaryotic BPL (Chapter 6). The work has been summarised by a general discussion and future directions for the project (Chapter 7). / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Candida parapsilosis is an emerging human opportunistic pathogen causing a wide spectrum of potentially life-threatening infections in immunocompromised hosts. One of the most important virulence factors of Candida spp. is a production of secreted aspartic proteinases (Saps). Presented thesis is mainly focused on the study of secreted aspartic proteinase 1 (Sapp1p) of C. parapsilosis, its processing and secretion under variable conditions and by use of various experimental models. Sapp1p is secreted by C. parapsilosis cells into the extracellular space as a completely processed and fully active enzyme. Experiments studying the C. parapsilosis cell wall (CW) confirmed the prolonged presence of completely processed Sapp1p on the cell surface (CW- Sapp1p). Proteolytic activity assay performed with the intact cells showed that CW-Sapp1p is proteolytically active prior to its release into the extracellular space and is capable of substrate cleavage. Biotinylation experiments with consecutive MS analysis revealed that CW-Sapp1p biotinylation is incomplete but saturable process, leaving partially unlabelled molecules. The accessibility of individual lysine residues in the Sapp1p molecule varied, with exception of four residues that were labelled in all of our experiments performed. The final step of...

A bacterial and archaeal adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), has recently been engineered for genome editing. This RNA-guided platform has simplified genetic manipulation and holds promise for therapeutic applications. However, off-target editing has been one of the major concerns of the commonly used Streptococcus pyogenes Cas9 (SpyCas9). Despite extensive enzyme engineering to reduce off-target editing of SpyCas9, we have turned to nature and uncovered a Cas9 ortholog from Neisseria meningitidis (Nme) with high fidelity. In the first part of my thesis, we have systematically characterized Nme1Cas9 for engineering mammalian genomes and demonstrated its high specificity by genome-wide off-targeting detection methods in vitro and in cellulo, and thus provided a new platform for accurate genome editing. Due to its flexibility, CRISPR is becoming a versatile tool not only for genome editing, but also for chromatin manipulation. These alternative applications are possible because of the programmable targeting capacity of catalytically dead Cas9 (dCas9). In the second part of my thesis, we have combined dCas9 with the engineered plant enzyme ascorbate peroxidase (APEX2) to develop a proteomic method called dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST). Relying on the spatially restricted, fast biotin labeling of proteins near defined genomic loci, C-BERST enables the high-throughput identification of known telomere- and centromere- associated proteomes and novel factors. Furthermore, we have extended C-BERST to map the c-fos promoter and gained new insights regarding the dynamic transcriptional regulation process. Taken together, C-BERST can advance our understanding of chromatin regulators and their roles in nuclear and chromosome biology.

L’interleukine 6 (IL-6) est une cytokine qui joue un rôle essentiel dans l’inflammation. Son récepteur (IL-6R) est composé de la chaîne non signalétique IL-6Rα et de la chaîne transductrice du signal gp130, commune aux cytokines de la famille IL-6. La liaison de l’IL-6 à son récepteur permet l’activation de plusieurs voies de signalisation, notamment des voies Jak/STAT1 et préférentiellement Jak/STAT3. De façon complémentaire, nous avons démontré que l’IL-6 est capable d’activer la voie Jak/STAT5 dans les lymphocytes T CD4. L’activation de cette voie de signalisation pourrait être impliquée dans le rétrocontrôle des effets pro-inflammatoires de l’IL-6 sur les cellules T CD4. Le facteur neurotrophique ciliaire (CNTF) et la « cardiotrophin-like cytokine/cytokine-like factor 1 » (CLC/CLF) sont deux cytokines de la famille de l’IL-6 qui signalent à travers un récepteur commun, le récepteur au CNTF (CNTFR), composé du CNTFRα, « leukaemia inhibitory factor receptor β » (LIFRβ) et gp130. Toutes deux exercent des actions au niveau du système immunitaire, or la chaîne CNTFRα de leur récepteur n’y est pas exprimée. Il a été montré que le CNTFR humain peut également activer un récepteur formé des sous-unités IL-6Rα, LIFRβ et gp130. Nous avons comparé les effets du CNTF et du CLC/CLF de souris sur des transfectants exprimant LIFRβ et gp130 et les chaines α connues de la famille IL-6 (IL-6Rα, IL-11Rβ et CNTFRα). Nos résultats indiquent que le CNTF de souris, comme le CNTF humain est capable d’activer un récepteur formé de l’IL-6Rα, LIFRβ et gp130. Toutefois cette propriété n’est pas partagée par CLC/CLF et le récepteur impliqué dans les effets de cette cytokine sur le système immunitaire reste donc à identifier. L’IL-27 appartient à la famille de l’IL-6 composée d’une sous-unité cytokinique, p28, associée à un récepteur soluble « l’Epstein-Barr virus-induced gene 3» (EBI3). La sous-unité p28 peut s’associer avec le récepteur soluble CLF pour former une cytokine capable d’activer les lymphocytes T. Dans le but de caractériser cette cytokine, nous avons montré que p28/CLF agit aussi sur les lymphocytes B et permet leur différenciation en plasmocytes. Le partage de l’IL-6R par l’IL-6 et p28/CLF semble être à l’origine de la similarité des effets de ces deux cytokines. De plus, nous avons observé des effets semblables à ceux de l’IL-6 suite à l’association de la sous-unité p28 seule avec la chaîne IL-6Rα. En effet, afin de mieux caractériser la cytokine p28/CLF, nous avons étudié les effets dus au recrutement de la chaîne IL-6Rα par la sous-unité p28. Les cytokines de la famille de l’IL-6 sont composées de quatre hélices α disposées de façon anti-parallèle deux à deux. La sous-unité p28 possède, au niveau d’une boucle reliant deux hélices α, un motif de plusieurs acides glutamiques consécutifs (motif polyE) qui n’est retrouvé dans aucune autre cytokine de cette famille. Nous avons démontré que ce motif est impliqué dans la liaison de cette sous-unité avec l’hydroxyapatite et l’os. Cette caractéristique de p28 pourrait permettre un ciblage de l’IL-27 (p28/EBI3) et de p28/CLF préférentiellement vers la niche endostéale des cellules souches et des cellules immunitaires. / Interleukin 6 (IL-6) is a well known cytokine, characterized for its essential function in inflammation. IL-6 receptor (IL-6R) is composed of IL-6Rα, an unsignalling chain, associated with the signaling transducing chain gp130. This glycoprotein is shared by all IL-6 family cytokines. After binding with its receptor, IL-6 preferentially induces the activation of the Jak/STAT3 pathway but can also activate the Jak/STAT1 pathway. Unexpectedly we demonstrated that IL-6 can activate the Jak/STAT5 pathway in CD4 T cells. This STAT5 could act as negative feedback mechanism in response to the pro-inflammatory effects induced by an excess of IL-6. Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC/CLF) both belong to the IL-6 cytokine family and share the same receptor, the CNTF receptor (CNTFR). CNTFR is composed of CNTFRα, leukaemia inhibitory factor receptor β (LIFRβ) and the glycoprotein gp130. Interestingly, the CNTFRα chain is not expressed by immune cells even though CNTF and CLC/CLF are active on these cells. These effects can be due to the formation of a complex between cytokine and CNTFRα, which can be shedded. This complex can then activate cells expressing only gp130 and LIFRβ. In human, it has been demonstrated that the CNTFRα chain can be substitute with IL-6Rα. Here, we compare mouse CNTF- and CLC/CLF-induced effects in transfected cells expressing LIFRβ, gp130 and different α chains belonging to the IL-6 family (IL-6Rα, IL-11Rα or CNTFRα). Our data demonstrate that like human CNTF, mouse CNTF is able to activate a receptor comprising of IL-6Rα, gp130 and LIFRβ. However, this property is not shared with CLC/CLF. Therefore, second receptor for this cytokine within the immune system still remains to be identify. Interleukin 27 (IL-27) belongs to the IL-6 cytokine family and is composed of the cytokine subunit p28 associated with a soluble receptor chain Epstein-Barr virus-induced gene 3 (EBI3). We demonstrate that the p28 subunit can bind the soluble receptor CLF to form a new dimeric cytokine named p28/CLF. This cytokine is active on T cells and our study demonstrates its activity on B cells. Our results show that p28/CLF sustains plasma cell differentiation. Those IL-6-like properties can be explained by the use of a common receptor, IL-6R. Moreover, our findings demonstrate that p28 has IL-6-like properties when associated with IL-6Rα. In order to better characterize p28/CLF, we next studied effects of to the recruitment of the IL-6Rα chain by p28 subunit. Cytokines belonging to the IL-6 family share a structural particularity by forming a four helix bundle cytokines family. The p28 subunit uniquely expresses a motif composed of a dozen of glutamic acids (polyE motif). We demonstrate that this motif permits p28 binding to hydroxyapatite and bone matrix. This observation could allow a preferential targeting to bone of IL-27 (p28/EBI3) and p28/CLF, and specifically a targeting of stem or immune cells to endosteal niches.

L’interleukine 6 (IL-6) est une cytokine qui joue un rôle essentiel dans l’inflammation. Son récepteur (IL-6R) est composé de la chaîne non signalétique IL-6Rα et de la chaîne transductrice du signal gp130, commune aux cytokines de la famille IL-6. La liaison de l’IL-6 à son récepteur permet l’activation de plusieurs voies de signalisation, notamment des voies Jak/STAT1 et préférentiellement Jak/STAT3. De façon complémentaire, nous avons démontré que l’IL-6 est capable d’activer la voie Jak/STAT5 dans les lymphocytes T CD4. L’activation de cette voie de signalisation pourrait être impliquée dans le rétrocontrôle des effets pro-inflammatoires de l’IL-6 sur les cellules T CD4. Le facteur neurotrophique ciliaire (CNTF) et la « cardiotrophin-like cytokine/cytokine-like factor 1 » (CLC/CLF) sont deux cytokines de la famille de l’IL-6 qui signalent à travers un récepteur commun, le récepteur au CNTF (CNTFR), composé du CNTFRα, « leukaemia inhibitory factor receptor β » (LIFRβ) et gp130. Toutes deux exercent des actions au niveau du système immunitaire, or la chaîne CNTFRα de leur récepteur n’y est pas exprimée. Il a été montré que le CNTFR humain peut également activer un récepteur formé des sous-unités IL-6Rα, LIFRβ et gp130. Nous avons comparé les effets du CNTF et du CLC/CLF de souris sur des transfectants exprimant LIFRβ et gp130 et les chaines α connues de la famille IL-6 (IL-6Rα, IL-11Rβ et CNTFRα). Nos résultats indiquent que le CNTF de souris, comme le CNTF humain est capable d’activer un récepteur formé de l’IL-6Rα, LIFRβ et gp130. Toutefois cette propriété n’est pas partagée par CLC/CLF et le récepteur impliqué dans les effets de cette cytokine sur le système immunitaire reste donc à identifier. L’IL-27 appartient à la famille de l’IL-6 composée d’une sous-unité cytokinique, p28, associée à un récepteur soluble « l’Epstein-Barr virus-induced gene 3» (EBI3). La sous-unité p28 peut s’associer avec le récepteur soluble CLF pour former une cytokine capable d’activer les lymphocytes T. Dans le but de caractériser cette cytokine, nous avons montré que p28/CLF agit aussi sur les lymphocytes B et permet leur différenciation en plasmocytes. Le partage de l’IL-6R par l’IL-6 et p28/CLF semble être à l’origine de la similarité des effets de ces deux cytokines. De plus, nous avons observé des effets semblables à ceux de l’IL-6 suite à l’association de la sous-unité p28 seule avec la chaîne IL-6Rα. En effet, afin de mieux caractériser la cytokine p28/CLF, nous avons étudié les effets dus au recrutement de la chaîne IL-6Rα par la sous-unité p28. Les cytokines de la famille de l’IL-6 sont composées de quatre hélices α disposées de façon anti-parallèle deux à deux. La sous-unité p28 possède, au niveau d’une boucle reliant deux hélices α, un motif de plusieurs acides glutamiques consécutifs (motif polyE) qui n’est retrouvé dans aucune autre cytokine de cette famille. Nous avons démontré que ce motif est impliqué dans la liaison de cette sous-unité avec l’hydroxyapatite et l’os. Cette caractéristique de p28 pourrait permettre un ciblage de l’IL-27 (p28/EBI3) et de p28/CLF préférentiellement vers la niche endostéale des cellules souches et des cellules immunitaires. / Interleukin 6 (IL-6) is a well known cytokine, characterized for its essential function in inflammation. IL-6 receptor (IL-6R) is composed of IL-6Rα, an unsignalling chain, associated with the signaling transducing chain gp130. This glycoprotein is shared by all IL-6 family cytokines. After binding with its receptor, IL-6 preferentially induces the activation of the Jak/STAT3 pathway but can also activate the Jak/STAT1 pathway. Unexpectedly we demonstrated that IL-6 can activate the Jak/STAT5 pathway in CD4 T cells. This STAT5 could act as negative feedback mechanism in response to the pro-inflammatory effects induced by an excess of IL-6. Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC/CLF) both belong to the IL-6 cytokine family and share the same receptor, the CNTF receptor (CNTFR). CNTFR is composed of CNTFRα, leukaemia inhibitory factor receptor β (LIFRβ) and the glycoprotein gp130. Interestingly, the CNTFRα chain is not expressed by immune cells even though CNTF and CLC/CLF are active on these cells. These effects can be due to the formation of a complex between cytokine and CNTFRα, which can be shedded. This complex can then activate cells expressing only gp130 and LIFRβ. In human, it has been demonstrated that the CNTFRα chain can be substitute with IL-6Rα. Here, we compare mouse CNTF- and CLC/CLF-induced effects in transfected cells expressing LIFRβ, gp130 and different α chains belonging to the IL-6 family (IL-6Rα, IL-11Rα or CNTFRα). Our data demonstrate that like human CNTF, mouse CNTF is able to activate a receptor comprising of IL-6Rα, gp130 and LIFRβ. However, this property is not shared with CLC/CLF. Therefore, second receptor for this cytokine within the immune system still remains to be identify. Interleukin 27 (IL-27) belongs to the IL-6 cytokine family and is composed of the cytokine subunit p28 associated with a soluble receptor chain Epstein-Barr virus-induced gene 3 (EBI3). We demonstrate that the p28 subunit can bind the soluble receptor CLF to form a new dimeric cytokine named p28/CLF. This cytokine is active on T cells and our study demonstrates its activity on B cells. Our results show that p28/CLF sustains plasma cell differentiation. Those IL-6-like properties can be explained by the use of a common receptor, IL-6R. Moreover, our findings demonstrate that p28 has IL-6-like properties when associated with IL-6Rα. In order to better characterize p28/CLF, we next studied effects of to the recruitment of the IL-6Rα chain by p28 subunit. Cytokines belonging to the IL-6 family share a structural particularity by forming a four helix bundle cytokines family. The p28 subunit uniquely expresses a motif composed of a dozen of glutamic acids (polyE motif). We demonstrate that this motif permits p28 binding to hydroxyapatite and bone matrix. This observation could allow a preferential targeting to bone of IL-27 (p28/EBI3) and p28/CLF, and specifically a targeting of stem or immune cells to endosteal niches.

Study of biosensors has become an essential part of research in biotechnology. Biosensors as fast, portable, highly sensitive, and low-cost bioanalytical detection devices have been utilized in many fields of human activity. The first part of the presented work focuses on electrochemical biosensors for rapid environmental screening of herbicides as water pollutants. A sol-gel immobilization method for a photosystem II (PSII) complex is studied in order to enhance the sensitivity and the signal strength and stability of a PSII-based biosensor. Computer simulations of a PSII biosensor are employed with the aim to find out how the immobilization membrane properties influence the biosensor parameters. Newly developed immobilization by a thin-layer membrane based on the results of computer simulations and revised measurement protocols are presented. The second part of the work is devoted to synthesis and electrochemical detection of newly developed metal labels for electrochemical immunosensors. The synthesis of dendrimer-encapsulated silver nanoparticles and biorecognition properties of biotin-nanocomposite conjugates are discussed. For detection of synthesized labels, a microfluidic detector was manufactured and tested and different approaches to packing of a microfluidic chip employing polydimethylsiloxane (PDMS) were investigated. Newly designed microstructures for a microfluidic separator of magnetic beads (MBs) were studied by computer simulations. The separator was made and trapping of MBs for the further employment in MBs-based immunoassays are presented

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNFalpha) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNFalpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNFalpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNFalpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.

Class I PI3Ks are a family (α, β, δ and γ) of ubiquitous lipid kinases that can be activated by cell surface receptors to 3-phosphorylate PI(4,5)P2 (phosphatidylinositol(4,5)-bisphosphate) and generate the signalling lipid PI(3,4,5)P3. The PI(3,4,5)P3 signal then activates a diverse collection of effector proteins involved in regulation of cell migration, metabolism and growth. The importance of this network is evidenced by the relatively high frequency with which cancers acquire gain-of-function mutations in this pathway and huge efforts to make PI3K inhibitors to treat cancer. The canonical model describing these events suggests class I PI3Ks are activated at the plasma membrane and generate PI(3,4,5)P3 in the inner leaflet of the plasma membrane where its effectors are activated. The PI(3,4,5)P3 signal can be terminated directly, by the tumour-suppressor and PI(3,4,5)P3-3-phosphatase PTEN, or modified to a distinct PI(3,4)P2 signal, by SHIP-family 5-phosphatases. The PI(3,4)P2 is removed by INPP4-family 4-phosphatases. Published work has shown that PI(3,4,5)P3 signalling can also occur in endosomes and nuclei, however, there is very little data defining the intracellular distribution of endogenous class I PI3Ks that supports these ideas; this is as a result of technical problems such as; their very low abundance, poor antibody-based tools and artefacts generated by overexpression of PI3Ks. Past work has indicated that, in PTEN-null mouse models of prostate tumour progression, either PI3Kβ or PI3Ks α and β, have important roles. Furthermore, the cell types and mechanism involved remained unclear. Recent published work in the host laboratory had indicated that there is an unexpectedly large accumulation of PI(3,4)P2 in PTEN-null cells that might be an important part of its status as a major tumour suppressor. The explanation and prevalence of this observation was unclear but potentially a result of PTEN also acting as a PI(3,4)P2 3-phosphatase in vivo. MEFs were derived from genetically-modified mice expressing endogenous, AviTagged class I PI3K subunits and used in experiments to define the subcellular localisation of class I PI3Ks. We found that following stimulation with PDGF, class IA PI3K subunits were unexpectedly depleted from the adherent basal membrane, in contrast, p85α and p110α, but not p85β and p110β, accumulated transiently in the nucleus. Interestingly, p110β, but none of the other subunits, was constitutively localised in the nucleus. These results support the idea that class I PI3K and PI(3,4,5)P3 signalling occurs in the nucleus. In organoids derived from WT, PI3Kγ-null or PTEN-null mouse prostate, application of PI3K-selective inhibitors revealed that PI3Kα had a dominant role in generating PI(3,4,5)P3 in prostate epithelial cells. The levels of PI(3,4)P2 were also elevated substantially in PTEN-null, compared to WT prostate organoids, use of PI3K-selective inhibitors suggested that it was also generated by PI3Kα. These data were consistent with the idea that PTEN can act as a PI(3,4)P2 3-phosphatase. Surprisingly, raising the pH of the organoids medium dramatically increased accumulation of PI(3,4,5)P3 and PI(3,4)P2, although the cause of this effect was unclear, we hypothesised the pH of the local environment may influence signalling via class I PI3Ks.