Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages
ARF6 is transiently activated and regulates uptake of zymosan particles. (a) Live-cell imaging of RAW264 macrophages expressing ARF6-wt-CFP during the phagocytosis of zymosan. The elapsed time is indicated at the top. The insets show higher-magnification images of the indicated regions of the cells. Phase-contrast images are shown (upper panels). Scale bar: 5 μm. (b) RAW264 cells were incubated with zymosan for various times. Cell lysates were incubated with GST-GGA3. The proteins associated with GST-GGA3 were pulled down using glutathione-sepharose beads and analyzed by western blotting (top panel). The middle panel shows aliquots of total lysates. The density of the protein bands was measured. The relative protein band intensity of GTP-ARF6 was normalized to total ARF6 (GTP-bound plus GDP-bound forms). (c) RAW264 cells transiently expressing CFP, ARF6-wt-CFP, ARF6-Q67L-CFP, or ARF6-T27N-CFP were incubated with zymosan and then fixed. The efficiency of phagocytosis was calculated. The results are expressed as a percentage of control (untransfected) cells. The means ± SEM of three independent experiments are plotted. Student’s -test was used for statistical analysis. compared to CFP transfected cells.
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