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.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32888 |
| Date | 31 August 2012 |
| Creators | Silver, Kristen |
| Contributors | Harrison, Rene E. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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