Although phosphatidylinositol (PI) and phosphoinositides (PIPs) only comprise a small percentage of the inner leaflet of the plasma membrane, they mediate a large variety of signaling events. In previous studies, we have observed the absence of macroscopically discernible domains in mixtures of PI/PC and PI(4,5)P2/PC. The addition of cholesterol to these mixtures results in condensation of the monolayer and hence domain formation. To better mimic the ionic conditions and hydrogen bonding properties of the inner leaflet plasma membrane, we investigated in this study the effect of common inner leaflet plasma membrane lipids like phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI, on phosphoinositide domain behavior in the presence of cholesterol and/or bivalent cations.

Autophagy requires the biogenesis of autophagosomes (APs), which are large multilamellar vesicles that sequester cytoplasmic substrates and undergo a maturation process that ultimately leads to their fusion with lysosomes. Previous studies have suggested that local production of phosphatidylinositol-3-phosphate (PI3P) by class III phosphatidylinositol 3-kinase (PI3K) (i.e., Vps34) is required for AP biogenesis at specialized sites of the endoplasmic reticulum called "omegasomes". Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. To address this question, we generated Vps34 knock-out mouse embryonic fibroblasts (MEFs) and analyzed the impact of Vps34 deletion on autophagy in mammalian cells. Using a novel higher affinity 4x-FYVE finger PI3P-binding probe, we found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in the Vps34 knock-out MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis. In addition, we used a lipidomic approach to capture the lipid profile of cells in the presence and absence of Vps34 under steady-state and during starvation-induced autophagy. Lipidomics is an emerging powerful tool with the potential to identify new interconnected metabolic lipid networks as well as generate new hypotheses. Here, we identified a new relationship between Vps34 and cholesterol homeostasis. Additionally, we identified specific changes in lysolipids during autophagy. Lastly, we investigated whether the retromer complex plays a role in autophagy. Retromer is a protein complex that binds PI3P on the endosomal membrane and mediates retrograde trafficking of transmembrane proteins from the endosome to the trans-Golgi network. Recent studies have shown a downregulation of this complex associated with sporadic Alzheimer's disease (AD) and have demonstrated aberrant trafficking and processing of APP, a pathological feature of AD, as a result of retromer deficiency. Because retromer is important for maintaining endo-lysosomal system function, we hypothesized that it promote efficient autophagy and may contribute to the dysfunctional autophagy observed in AD when impaired. Using standard autophagy assays, such as assessing LC3 conjugation and puncta formation, our preliminary studies suggest a negative regulatory role for retromer in autophagy. Additionally, we observed a strong association of retromer with Atg9, an autophagy-related gene transmembrane protein that is believe to traffic lipids to the growing autophagosome membrane and recycle autophagy proteins from this compartment.

Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase (PI3K), is an essential mediator of the PI3K/AKT signalling pathway, which is crucial for cell proliferation, survival and apoptosis. Constitutive activation of this signalling cascade has been identified in acute myeloid leukaemia (AML), the most common haematopoietic malignancy in adults, and experimental deletion of the PI3K antagonists PTEN and SHIP cause leukaemia in mice. However, little is known regarding the role of other PI modulator proteins in AML. Thus, in this thesis, a lentivirally delivered small hairpin RNA (shRNA) library targeting 103 genes (345 pLKO knockdown constructs) with presumed or established roles in PI metabolism was utilised to screen for genes required for AML blast cell viability/proliferation and differentiation. First, knockdown constructs were tested for their impact on proliferation/viability in seven human AML cell lines by measuring fold change in fluorescence of the cell viability dye alamarBlue relative to controls (cells transduced with a non-targeting control hairpin) over three days. This identified 13 candidate genes selected with the criterion that two or more knockdown constructs per gene reduce cell viability/proliferation relative to control by greater than or equal to50 % across all cell lines. From these candidate genes, PIP4K2A, INPP5B and IMPAD1 were selected for downstream validation experiments, which reproduced the observation from the primary screen. For INPP5B and IMPAD1, knockdown constructs also reduced clonogenic potential of primary human AML samples but only showed a modest effect on normal CD34+ haematopoietic stem or progenitor cells (HSPCs) in a methylcellulose based assay. This could be recapitulated in a murine setting where knockdown constructs targeting both genes reduced clonogenic potential of murine MLL- AF9 AML cells with little effect on normal KIT+ HSPCs. In line with this, Inpp5b knockout KIT+ BM cells either failed to immortalise or weakly immortalised, following forced expression of the powerful MLL-AF9 oncogene. A further screen was performed to identify regulators of THP-1 blast cell differentiation, by seeding knockdown construct transduced cells into methylcellulose based semisolid media. After ten days of incubation the degree of macrophage differentiation was evaluated by light microscopy and an arbitrary differentiation score was given. With the criterion that greater than or equal to2 knockdown constructs per gene received the highest differentiation score, reflecting terminal macrophage differentiation of all seeded cells, SBF2 was identified as the top-scoring hit. Validation experiments have confirmed macrophage differentiation based on cytospin preparations of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have shown that knockdown constructs targeting PIP4K2A and SBF2 delayed or abrogated in vivo leukaemogenesis. Thus this work has identified novel roles for PI modulator genes in human AML with possible therapeutic potential.

Mycobacterial cell wall components have been shown to elicit a range of immunological responses in mammalian hosts. A family of cell wall antigens, the phosphatidylinositol mannans (PIMs), have been shown to reduce allergic response in a murine model of allergic airway disease and have been suggested as potential therapeutic agents. Isolation and characterisation of these compounds is not facile. To confirm the structure of PIMs a number of phosphatidylinositols (PIs), 1a-c, PIM1s 2a, 2d and 2e, and AcPIM1s, 2g and 2f, were prepared to allow assignment of the acylation pattern of natural products and for evaluation in immunological assays. As the natural products include 19:0 acylation in the form of (R)-tuberculostearoyl residues, a source of (R)-tuberculostearic acid was needed. To this end, an efficient synthesis of (R)-tuberculostearic acid from (S)-citronellol, utilising a copper-catalysed cross-coupling reaction and a modified Julia olefination, was developed. This material was incorporated into diacylglycerols prepared from (R)-benzyl glycidol. A protected myo-inositol derivative, 188, and two protected pseudo-disaccharides, 10 and 241, were prepared from myo-inositol via desymmetrisation utilising a camphylidene acetal. These were coupled with diacylglycerols via a phosphate ester and deprotected to give PIs, PIM1s and AcPIM1s. Mass spectrometry studies were undertaken on the PIs, 1a-c, PIM1s 2a, 2d and 2e, and AcPIM1s, 2g and 2f which structures that have been established by chemical synthesis. Comparison of these data with those reported for natural PIs and PIMs containing 19:0 ((R)-tuberculostearoyl) and 16:0 (palmitoyl) acyl groups unequivocally established that the 19:0 residue was located at the sn-1 and the 16:0 at the sn-2 position of the glycerol moiety in nature.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

Thesis (Ph.D.) - University of St Andrews, January 2008. / Restricted until 15th January 2009, during which time access is available with the consent of the Head of School.

Thesis (MSc)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (Ph. D. in Pharmacology)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Le metabolisme des phosphoinositides est constitue dfun reseau complexe dfenzymes et de seconds messagers participant a la regulation de nombreux processus cellulaires :la proliferation, la croissance, la survie et la migration. Le phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), est un second messager intracellulaire tres important dont le taux est controle a la fois au niveau de sa synthese par des kinases et au niveau de sa degradation par des phosphatases. La phosphatase PTEN (phosphatase and tensin homolog deleted on chromosomes 10), gene suppresseur de tumeurs frequemment mutee dans des cancers (comme dans des glioblastomes), le dephosphoryle en position 3. Il existe egalement une activite á inositol 5-phosphatase â pour de nombreux derives du myo-inositol. Cfest la proteine SHIP2 (SH2-containing Inositol 5-phosphatase 2), une phosphatase membre de la famille des phosphatidylinositol polyphosphate 5-phosphatases qui est, entre autre, responsable de cette activite.Le but principal de ce travail de these a ete de mettre en evidence le role pro ou anti-oncogenique de SHIP2 dans un modele cellulaire humain de glioblastomes :les cellules 1321 N1. Ce modele cellulaire a comme particularite lfabsence dfexpression de PTEN au niveau proteique.La caracterisation des clones cellulaires deficients pour SHIP2 nous a permis de mettre en evidence une augmentation du taux du PtdIns(3,4,5)P3 par rapport a des cellules qui expriment un taux normal de SHIP2. Par un simple comptage, nous avons observe une augmentation du nombre de cellules en culture par rapport aux cellules controles. Lfimmunomarquage de la F-actine nous a permis de montrer une difference de morphologie entre les cellules N1 shSHIP2 et les cellules controles. Au travers de Western blots, nous avons observe que la phosphorylation de la PKB ainsi que celle de Erk1/2 etait augmentee dans les cellules deficientes pour SHIP2 en comparaison aux cellules qui expriment SHIP2.Dans les cellules COS-7 transfectees par SHIP2 sauvage, nous avons identifie par spectrometrie de masse 8 sites de phosphorylation sur Tyr, Ser et Thr. En systeme endogene dans les cellules N1, notre etude a demontre la presence de 3 sites de phosphorylations sur Ser (position 132 et 1258) et Thr (position 1254). Nous avons genere deux anticorps specifiques contre la proteine SHIP2 phosphorylee sur la Ser132 et Thr1254. Lfanticorps contre la Ser 132 nous a permis de mettre en evidence une colocalisation entre pSHIP2 Ser132 et le facteur dfepissage SC35 present dans les speckles nucleaires. Cette donnee nous suggere un role nucleaire nouveau pour SHIP2.De maniere originale nous avons mis en evidence dans les cellules dfastrocytomes N1 trois localisations subcellulaires de SHIP2 :i) a la membrane plasmique au niveau des adherences focales ou SHIP2 serait impliquee dans le controle du taux de PtdIns(3,4,5)P3/ PtdIns(4,3)P2, et de la migration ii) au niveau perinucleaire ou SHIP2 ainsi que la forme phosphorylee sur Ser132 seraient impliquees dans des interactions proteiques via ses proprietes de á docking protein â et enfin iii) dans le noyau specifiquement dans les speckles ou la forme phosphorylee de SHIP2 sur Ser132 serait impliquee dans le controle du taux de PtdIns(4,5)P2, mais egalement dans la signalisation nucleaire impliquant les phosphoinositides.La presence de SHIP2 dans les adherences focales nous a amene a rechercher par quel mecanisme SHIP2 pouvait se retrouver a cette localisation. Nous avons par immunoprecipitation suivie dfune analyse par spectrometrie de masse identifie la myosine 1c (Myo1c) comme nouveau partenaire de SHIP2. Les cellules deficientes pour la Myo1c presentent une modification de la morphologie cellulaire, ainsi qufune modification de la localisation intracellulaire de SHIP2. Les cellules N1 deficientes pour la Myo1c montrent aussi une diminution de lfexpression de la sous unite regulatrice de la PI 3-kinase (p85ƒ¿) ainsi qufune absence de la phosphorylation de la PKB sur la Ser473 et la Thr308. En outre, ces cellules N1 shMyo1c presentent un taux eleve dfapoptose compare aux cellules controles.En conclusion, ces travaux ont permis de montrer que dans un modele humain de glioblastomes N1, SHIP2 etait phosphorylee sur la Ser 132 et que cette phosphorylation semblait etre requise pour son activite phosphatase. SHIP2 possedait trois localisations subcellulaires probablement associees a des fonctions differentes, qufelle interagissait au minimum avec les proteines partenaires nouvelles lamin A/C et Myo1c. Enfin SHIP2 exercait un controle negatif sur la proliferation et la migration de ces cellules. Ces resultats mettent en evidence plusieurs fonctions originales de SHIP2 comme son role dans la migration et la proliferation qui ouvrent des perspectives nouvelles dans lfetude de cette phosphatase dans le contexte general et surtout physiopathogique des fonctions attribuees aux phosphoinositides. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished