title: Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules.
abstract: Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.
authors.affiliation: Department of Biological Sciences, University of Toronto at Scarborough, Toronto, Ontario, Canada.
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Binker
authors.forename: Dorothy Y
authors.initials: DY
authors.lastname: Zhao
authors.forename: Sophie J Y
authors.initials: SJ
authors.lastname: Pang
authors.forename: Rene E
authors.initials: RE
authors.lastname: Harrison
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects / Q000276:immunology*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000276:immunology*; D017737:Macrophages, Peritoneal / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000502:physiology*; D019319:Okadaic Acid / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects / Q000276:immunology*; D016192:Resting Phase, Cell Cycle / Q000187:drug effects / Q000276:immunology; D000099163:Myosin Binding Protein C