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

Figure 6

Plasma membrane-targeted ACAP2 inhibits zymosan uptake through ARF6 inactivation. (a) Quantification of zymosan phagocytosis in cells expressing several CFP-ACAP2 mutants was conducted. The number of formed phagosomes was counted under the microscope. The efficiency of phagocytosis was calculated based on 50 transfected and 50 untransfected cells. The results are expressed as a percentage of control (untransfected) cells. The data are means ± SEM of three independent experiments. It is of note that the expression of plasma membrane-targeted ACAP2 dramatically reduces phagosome formation. Student’s -test was used for statistical analysis. compared to Mem-CFP. (b) Levels of endogenous ARF6 activation were determined in RAW264 cells expressing Mem-CFP-ACAP2 or Mem-CFP-ACAP2-RQ. Cell lysates were prepared and incubated with GST-GGA3. The proteins associated with GST-GGA3 were pulled down using glutathione-sepharose beads. GTP-ARF6 (top panel), total ARF6 (second panel), and CFP fusion proteins (third panel) were detected by immunoblotting. GST-GGA3 was stained with Ponceau S (bottom panel). Note that the expression of plasma membrane-targeted ACAP2 impairs ARF6 activation. Similar results were obtained from three independent experiments.

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