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Journal of Immunology Research
Volume 2015, Article ID 429439, 12 pages
http://dx.doi.org/10.1155/2015/429439
Research Article

Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

1Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan
2Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan

Received 10 February 2015; Accepted 25 May 2015

Academic Editor: Ghislain Opdenakker

Copyright © 2015 Youhei Egami et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.