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Cytoplasmic Linker Protein-170 Enhances Spreading and Phagocytosis in Activated Macrophages by Stabilizing Microtubules¹

Marcelo G. Binker^*, Dorothy Y. Zhao^*, Sophie J. Y. Pang and Rene E. Harrison^2,*,

^* Department of Biological Sciences, University of Toronto at Scarborough, Toronto, Ontario, Canada; and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

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- and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated -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-/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-/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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Canadian Institutes for Health Research Grant MOP-68992 and a Natural Science and Engineering Research Council grant to R.E.H. D.Y.Z. is the recipient of a Natural Science and Engineering Research Council Undergraduate Student Research Award. S.J.Y.P. is the recipient of a University of Toronto fellowship award. R.E.H. is the recipient of the Ontario Early Researcher Award.

² Address correspondence and reprint requests to Dr. Rene E. Harrison, Department of Biological Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto, Ontario, M1C 1A4, Canada. E-mail address: harrison{at}utsc.utoronto.ca

³ Abbreviations used in this paper: MT, microtubule; CLIP-170, cytoplasmic linker protein-170; DN, dominant negative; OA, okadaic acid; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; RPM, resident peritoneal macrophage; +TIP, MT plus-end tracking protein; WT, wild type; DIC, differential interference contrast.

⁴ The online version of this article contains supplemental material.