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viii, 139, xii leaves : ill. ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1974

No description available.

No description available.

Pseudopod formation during phagocytosis is a limiting step in managing the uptake of particles and here we show that the conventional kinesin is involved in both receptor and membrane delivery to the phagocytic cup. Expression of a mutant kinesin isoform (EGFP-Kif5B-DN) in RAW264.7 cells significantly reduced binding of IgG-sRBCs when macrophages were faced with multiple encounters with opsonized particles. SEM analysis of EGFP-Kif5B-DN expressing cells showed sparse, extremely thin pseudopods. We saw disrupted Rab11 trafficking to the phagocytic cup in EGFP-Kif5B-DN-transfected cells. Our opsonized particle overload assays also implicated phagosome membrane recycling in pseudopod formation. We observed reduced phagosome fission and trafficking in mutant kinesin expressing cells as well as reduced cell surface expression of Fcy and Mac-1 receptors. It is evident that anterograde trafficking via kinesin is essential both for receptor recycling from the phagosome as well as delivery of Rab11-containing membrane stores to effect broad and functional pseudopods. We noticed an actin cup defect in EGFP-Kif5B-DN-transfected cells and turned to focal adhesions to investigate the role of kinesin in actin assembly at the membrane. To investigate the role for kinesin in focal adhesion formation we examined nascent focal adhesions formation in CHO-ΙΙA cells. Transfected, EGFP-Kif5B-DN transfected cells were less spread compared to control cells and displayed a “halo” pattern of focal adhesions at the base of the cell. To see if this was a defect in upstream receptors involved in focal adhesion assembly we investigated surface integrin levels in EGFP-Kif5B-DN transfected cells. Using flow cytometry and immunofluorescence analysis we observed decreased levels of β1-integrin at the surface of EGFP-Kif5B-DN transfected cells, compared to control cells. We investigated the signaling requirements for kinesin-mediated integrin transport and determined that β1-integrin transport from the recycling compartment is regulated by the PI(3)K-PKB/AKT-GSK-3β pathway. Thus in the absence of functional kinesin, cells fail to form proper focal adhesion sites due to lack of adhesion and substrate contact resulting in the loss of cell spreading.

Pseudopod formation during phagocytosis is a limiting step in managing the uptake of particles and here we show that the conventional kinesin is involved in both receptor and membrane delivery to the phagocytic cup. Expression of a mutant kinesin isoform (EGFP-Kif5B-DN) in RAW264.7 cells significantly reduced binding of IgG-sRBCs when macrophages were faced with multiple encounters with opsonized particles. SEM analysis of EGFP-Kif5B-DN expressing cells showed sparse, extremely thin pseudopods. We saw disrupted Rab11 trafficking to the phagocytic cup in EGFP-Kif5B-DN-transfected cells. Our opsonized particle overload assays also implicated phagosome membrane recycling in pseudopod formation. We observed reduced phagosome fission and trafficking in mutant kinesin expressing cells as well as reduced cell surface expression of Fcy and Mac-1 receptors. It is evident that anterograde trafficking via kinesin is essential both for receptor recycling from the phagosome as well as delivery of Rab11-containing membrane stores to effect broad and functional pseudopods. We noticed an actin cup defect in EGFP-Kif5B-DN-transfected cells and turned to focal adhesions to investigate the role of kinesin in actin assembly at the membrane. To investigate the role for kinesin in focal adhesion formation we examined nascent focal adhesions formation in CHO-ΙΙA cells. Transfected, EGFP-Kif5B-DN transfected cells were less spread compared to control cells and displayed a “halo” pattern of focal adhesions at the base of the cell. To see if this was a defect in upstream receptors involved in focal adhesion assembly we investigated surface integrin levels in EGFP-Kif5B-DN transfected cells. Using flow cytometry and immunofluorescence analysis we observed decreased levels of β1-integrin at the surface of EGFP-Kif5B-DN transfected cells, compared to control cells. We investigated the signaling requirements for kinesin-mediated integrin transport and determined that β1-integrin transport from the recycling compartment is regulated by the PI(3)K-PKB/AKT-GSK-3β pathway. Thus in the absence of functional kinesin, cells fail to form proper focal adhesion sites due to lack of adhesion and substrate contact resulting in the loss of cell spreading.

The aim of this project was to investigate the role of lipid rafts in actin-mediated phagocytosis. Lipid rafts are defined as highly condensed membrane domains enriched in cholesterol and glycosphingolipids and are thought to participate in a range of cellular functions including actin-mediated phagocytosis. Remodelling of the actin skeleton facilitates the formation of a phagocytic membrane cup and drives the uptake of particles. Hence, actin restructuring is essential for phagocytosis. How engagement of Fc receptors triggers membrane re-organization at the site of phagocytosis and how the formation of ordered raft domains is linked to actin remodelling during phagosome maturation is currently not known. Lipid rafts potentially form platforms for local signal transduction for Fc surface receptors and secondary messengers. Raft distribution therefore would critically influence and direct their function. The hypothesis is that lipid rafts are the membrane sites on the cell surface, which enable, drive and localise actin- dependent phagocytosis. Phagocytosis in macrophages was induced with IgG-coated beads of different sizes as substrates for uptake via the Fc receptor mediated pathway. Membrane order was visualised and quantified by two-photon microscopy. Actin remodelling was imaged in parallel with confocal microscopy. Time-course and live cell imaging demonstrated that phagocytosis induces formation of highly ordered membrane domains around the phagocytic particle independently of the particle size. The high membrane order is the biophysical hallmark of lipid rafts suggested that Fc receptor cross-linking induces the coalescence of lipid rafts. Live-cell imaging further identified a temporal correlation between membrane condensation and actin restructuring at sites of phagocytosis. Membrane condensation persisted after actin detached from the sealed phagosome. Receptor clustering induced by particle binding activates Src kinases leading to tyrosine phosphorylation of ITAM motif of the receptors, activation of GTPases and actin polymerisation. Lipid raft recruitment may be driven by these events or alternatively, rafts be essential for kinase activation. Several inhibitors were used to interrupt crucial steps in the signalling cascade leading to actin restructuring. Laurdan microscopy showed that membrane order is independent of Lyn activation (inhibited with PP2), PI3K activity (inhibited with Wortmannin) and actin polymerisation (Latrunculin B). Inhibitors had differential effects on phagocytosis rates of small and large particles. Inhibition of Lyn had a more severe effect on phagocytosis of large beads than on phagocytosis of small beads. Disruption of PI3K activity with Wortmannin only inhibited phagocytosis of large but not of small particles, whereas disruption of the actin skeleton with Latrunculin B inhibited phagocytosis of small and large particles. These data suggest that membrane condensation is independent of kinase activity and occurs upstream of actin remodelling. The role of lipid rafts in phagocytosis was further investigated by modulation of sterol composition of the cell membranes. Cholesterol depletion with methyl-- cyclodextrin disrupted membrane organization at phagosomes of small and large beads and also abolished phagocytosis. However, the fluidity of the entire plasma membrane was increased upon treatment of cells with methyl--cyclodextrin suggesting that this condition was not specifically affecting phagosomal membranes. Cholesterol enrichment increased membrane condensation even further than the membrane condensation detected in control phagosomes. Incorporation of 7-keto-cholesterol (7KC) decreased membrane order of phagosomes of small beads but not of large beads. 7KC can prevent membrane condensation due to its additional keto-group, which acts as a spacer between phospholipids. Phagocytosis of large beads but not of small beads was affected by 7KC incorporation. This suggests that 7KC only moderately reduces membrane order, which diminishes but not completely abolishes phagocytosis. This might be explained by the fact that 7KC enrichment and mCD treatment diminished actin remodelling and reduced the complexity of the F-actin network. Mass spectrometry was employed to quantify the lipidome of phagosomal membranes. This is the first study that directly demonstrates that phagosomes exhibit a distinct lipid composition and were enriched in sphingomyelin (SM) but depleted of cholesterol. Furthermore, the effects of sterol modulation on lipid species abundance were investigated. Cholesterol and 7KC enrichment resulted in lower levels of PC, but higher levels of charged lipids. In addition, 7KC treatment increased SM levels. In conclusion, cross-linking of Fc receptors triggers the formation of ordered membrane domains that do not have the classical raft composition. They are cholesterol depleted but rich in sphingomyelin. The formation of these ??rafts?? occurs upstream of actin remodelling and is necessary for actin remodelling during phagocytosis.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology, 1974.