title: Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages.
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.
authors.affiliation: Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
authors.forename: Youhei
authors.initials: Y
authors.lastname: Egami
authors.affiliation: Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Fujii
authors.affiliation: Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Kawai
authors.affiliation: Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
authors.forename: Yurie
authors.initials: Y
authors.lastname: Ishikawa
authors.affiliation: Laboratory 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.
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Araki
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D058948:Cell Tracking / Q000379:methods; D020690:GTPase-Activating Proteins / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000378:metabolism; D015054:Zymosan / Q000276:immunology*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C570848:ACAP2 protein, mouse; D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D015054:Zymosan; C543587:Rab35 protein, mouse; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse; D020691:rab GTP-Binding Proteins