Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages
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...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2015-01-01
|
| Series: | Journal of Immunology Research |
| Online Access: | http://dx.doi.org/10.1155/2015/429439 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849410915177332736 |
|---|---|
| author | Youhei Egami Makoto Fujii Katsuhisa Kawai Yurie Ishikawa Mitsunori Fukuda Nobukazu Araki |
| author_facet | Youhei Egami Makoto Fujii Katsuhisa Kawai Yurie Ishikawa Mitsunori Fukuda Nobukazu Araki |
| author_sort | Youhei Egami |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-61084ef7f88e4f8b9f571b45355fa855 |
| institution | Kabale University |
| issn | 2314-8861 2314-7156 |
| language | English |
| publishDate | 2015-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Immunology Research |
| spelling | doaj-art-61084ef7f88e4f8b9f571b45355fa8552025-08-20T03:34:56ZengWileyJournal of Immunology Research2314-88612314-71562015-01-01201510.1155/2015/429439429439Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 MacrophagesYouhei Egami0Makoto Fujii1Katsuhisa Kawai2Yurie Ishikawa3Mitsunori Fukuda4Nobukazu Araki5Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, JapanDepartment of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, JapanDepartment of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, JapanDepartment of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, JapanLaboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, JapanDepartment of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, JapanPhagocytosis 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.http://dx.doi.org/10.1155/2015/429439 |
| spellingShingle | Youhei Egami Makoto Fujii Katsuhisa Kawai Yurie Ishikawa Mitsunori Fukuda Nobukazu Araki Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages Journal of Immunology Research |
| title | Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages |
| title_full | Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages |
| title_fullStr | Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages |
| title_full_unstemmed | Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages |
| title_short | Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages |
| title_sort | activation inactivation cycling of rab35 and arf6 is required for phagocytosis of zymosan in raw264 macrophages |
| url | http://dx.doi.org/10.1155/2015/429439 |
| work_keys_str_mv | AT youheiegami activationinactivationcyclingofrab35andarf6isrequiredforphagocytosisofzymosaninraw264macrophages AT makotofujii activationinactivationcyclingofrab35andarf6isrequiredforphagocytosisofzymosaninraw264macrophages AT katsuhisakawai activationinactivationcyclingofrab35andarf6isrequiredforphagocytosisofzymosaninraw264macrophages AT yurieishikawa activationinactivationcyclingofrab35andarf6isrequiredforphagocytosisofzymosaninraw264macrophages AT mitsunorifukuda activationinactivationcyclingofrab35andarf6isrequiredforphagocytosisofzymosaninraw264macrophages AT nobukazuaraki activationinactivationcyclingofrab35andarf6isrequiredforphagocytosisofzymosaninraw264macrophages |