Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules.
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.
Binker
Marcelo G
MG
Department of Biological Sciences, University of Toronto at Scarborough, Toronto, Ontario, Canada.
Zhao
Dorothy Y
DY
Pang
Sophie J Y
SJ
Harrison
Rene E
RE
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