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.

Thesis (M.S.)--Missouri University of Science and Technology, 2008. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed January 22, 2009) Includes bibliographical references (p. 34-40).

Thesis (Ph. D.)--State University of New York at Binghamton, Dept. of Chemistry, 2007.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2002. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Kenneth A. Johnson. Vita. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Th.--Biol. cell.--Toulouse 3, 1983. N°: 1100.

All cells contain cytoskeletal components which perform static 'spatial organizing' and dynamic 'motile' functions. The studies described in this thesis concern two types of cytoskeletal component of universal biological importance: microtubules and microfilaments, and various other structures which are associated with them. The general properties of microtubules and microfilaments and of other cytoskeletal components are reviewed in the introduction. Examples of the functions they perform in various systems are described, with emphasis on Protozoa and metazoan morphogenetic systems. The ciliate Nassula aurea was the first system studied. The cytopharyngeal basket is a tube shaped feeding organelle through which rapid cytoplasmic streaming occurs. The basket becomes deformed during transport of a food vacuole through its lumen. Ultrastructural examination of deformed baskets reveal that part of the basket, the tubular 'sheath', becomes stretched, probably in an elastic manner. Links between the microtubules in the sheath also appear to become stretched, so these could be responsible for the elastic properties. The nature of the links between microtubules within the 'rods', components of the basket responsible for its longitudinal rigidity, is also discussed, as are some other features relevant to the function of the basket as a feeding organelle. The second system studied was oogenesis in Drosophila melanogaster. Several stages in the development of the oocyte and the surrounding follicle cells and nurse cells were examined ultrastructurally. The arrangement of microtubules and microfilaments is described, as are the changes that occur in these and other structures during oogenesis, and the related changes in morphology. A circumferentially oriented layer of microfilaments was discovered in vitellogenic oocyte follicles. The oocyte expands rapidly during vitellogenesis, as cytoplasm flows into it from the nurse cells, and as yolk proteins and nutrients flow in across the follicle. The microfilamentous layer could cause the oocyte to elongate as it expands by restricting its increase in diameter. The layer is destroyed by cytochalasin B, and can be decorated by heavy meromyosin. Therefore it is concluded that the microfilaments are actin-like. Various other structures in the developing egg chamber (the oocyte plus follicle and nurse cells) are described.