Effects of overexpression of ARF isoforms on FcγR-mediated phagocytosis. (A) Phagocytic activities of P388D1 cells against IgG-opsonized and nonopsonized polystyrene beads. P388D1 cells, preincubated with (+) or without (−) anti–mouse FcγRII/III mAb 2.4G2 for 30 min at 4°C, were incubated with polystyrene beads nonopsonized or opsonized with rabbit IgG for 20 min at 4°C, followed by 30 min at 37°C. After removal of unbound beads, the number of beads attached and ingested per single cell (the association index) was counted as described in Materials and Methods. (B and C) Inhibition of FcγR-mediated phagocytosis by overexpression of the class III ARF (ARF6), but not other classes of ARFs. P388D1 cells transiently transfected with the indicated constructs in pcDNA3 vectors were incubated with IgG-opsonized beads as above. Each ARF cDNA was tagged with HA tag. The number of beads ingested per single cell (the phagocytosis index) was determined for cells expressing the indicated constructs and for nonexpressing controls (MOCK), as described in Materials and Methods. Wild-type cDNAs of ARF1, ARF5, and ARF6 were used in B, and wild-type and Q67L and T27N mutants of ARF6 were used in C. The exogenous expression of HA-tagged ARFs, as detected by immunoblotting using anti-HA mAb, is shown below. The expression levels of exogenous wild-type ARF6-HA were 8.1 ± 0.5–fold higher than endogenous ARF6. This estimation was obtained by immunostaining the mock-transfected cells and cells expressing ARF6-HA using an anti-ARF6 mAb coupled with Cy3-anti–mouse IgG, and differences in fluorescent intensities of single cells in these two samples were compared as described in Materials and Methods. Abs that specifically recognize ARF1 or ARF5 were not available. (D and E) Overexpression of ARF isoforms does not affect the binding to IgG-opsonized beads and surface expression of FcγRs. Binding to IgG-opsonized beads was assessed by incubating cells with the beads for 20 min at 4°C. Unbound beads were then removed by washing, and numbers of beads attached to the transfected cells are shown (the attachment index) (D). Other procedures are as described above. (E) Surface levels of FcγRs on cells transiently transfected with the indicated ARF cDNA constructs and, hence, expressing the exogenous proteins were assessed by staining the nonpermeabilized cells with 2.4G2 mAb (solid line; see Materials and Methods). Control uses irrelevant rat IgG (broken line). In A–D, data are expressed as the mean ± SE of three independent experiments. *P < 0.001.

Overexpression of PAG3, but not its GAP-inactive mutant or GIT2s, attenuates the focal accumulation of F-actin beneath IgG-opsonized beads and its restoration by cooverexpression of ARF6. P388D1 cells and cells transiently transfected with the indicated plasmid constructs were incubated with IgG-opsonized beads and processed for laser confocal microscopic analysis, as described in the legend to Fig. 1, except that HA-tagged ARF6 was visualized with anti-HA mAb coupled with Cy5-anti–mouse IgG. All plasmid constructs were derived from pcDNA3 or pEGFP, and the overexpression of exogenous proteins was confirmed as in the legends to Fig. 2 and Fig. 3. (A) Cells were mock-transfected (MOCK; panels a–e), or transfected with plasmid DNAs for wild-type EGFP-PAG3 (panels f–j), its GAP-inactive mutant (panels k–o), wild-type HA-GIT2-short (panels p–t), and wild-type HA-GIT2 (panels u–y). Exogenous ARFGAP proteins (panels a, f, k, p, and u) and F-actin (panels b, g, l, q, and v) were visualized as described in the legend to Fig. 1. The third column of panels (c, h, m, r, and w) represent the merging of the first two columns of panels, and their higher magnifications are shown in the fourth column of panels (indicated by rectangles; panels d, i, n, s, and x). The last column of panels (e, j, o, t, and y) show the DIC images of the same fields of the left four panels. (B) Cells were cotransfected with 1.5 μg of plasmid DNA for wild-type EGFP-PAG3 together with 0.5 μg of plasmid DNA for ARF6-HA; and EGFP-PAG3 (panel a), ARF6-HA (panel b), and F-actin (panel c) were visualized as above (see also the legend to Fig. 5 C, third column). Panel d represents the merging of the three left panels. Panel e shows the DIC image of the same field of the first four panels. Arrows indicate the position of phagocytic beads. Bars, 5 μm.

PAG3, but not GIT2s, accumulates to phagocytic cups, together with F-actin and ARF6. P388D1 cells and cells transfected with pBabe/EGFP-PAG3, pBabe/HA-GIT2-short, or pBabe/HA-GIT2 were incubated with IgG-opsonized beads for 20 min at 4°C, and fixed after incubation at 37°C for 5 min. (A) Endogenous PAG3 (panel a) was visualized using an anti-PAG3 Ab coupled with Cy2-anti–rabbit IgG, and EGFP-PAG3 (panel e), HA-GIT2-short (panel i), and HA-GIT2 (panel m) were visualized by detecting the auto-fluorescence from the EGFP, or by staining cells with an anti-HA mAb coupled with Cy2-anti–mouse IgG. F-actin was visualized with Texas red X–conjugated phalloidin (panels b, f, j, and n). The third panels represent the merging of the left two panels. The right panels show the differential interference contrast (DIC) images of the same fields of the three preceding panels. Arrows indicate positions of phagocytic beads. The expression levels of EGFP-PAG3 in each cell were almost equal or only slightly higher compared with the levels of endogenous PAG3, as assessed by staining EGFP-PAG3–expressing cells with an anti-PAG3 Ab, coupled with Cy3-anti–rabbit IgG, and comparing their fluorescent signals with those of untransfected cells (the transfected cells gave 2.4 ± 0.1–fold higher signals than the untransfected cells; see Materials and Methods). (B) EGFP-PAG3 (panel a) and F-actin (panel c) were visualized as above; endogenous ARF6 was detected using anti-ARF6 mAb coupled with Cy5-anti–mouse IgG (panel b). Panel d represents the merging of panels a–c, and panel e shows the DIC images of the same fields of panels a–d. Higher magnifications of images of panels a–d (indicated by rectangles) are each shown below (panels f–i). Bars, 5 μm.

Overexpression of PAG3, but not its GAP-inactive mutant or GIT2s, inhibits FcγR-mediated phagocytosis. (A and B) P388D1 cells (MOCK) and cells transiently transfected with the empty pEGFP-C1 vector (Vec), or the plasmids containing cDNAs for wild-types (WT) or the GAP-inactive mutants (CA) of EGFP-PAG3, HA-GIT2, and HA-GIT2-short, were incubated with IgG-opsonized beads for 20 min at 4°C, followed by 30 min at 37°C to define the phagocytosis index (A); or only for 20 min at 4°C to define the attachment index (B). Cells expressing the exogenous proteins were examined as described in Materials and Methods. Data are expressed as the mean ± SE of three independent experiments. *P < 0.001. The exogenous expression of HA-tagged GIT2s, EGFP-tagged PAG3s and EGFP, detected by immunoblotting using anti-HA mAb and anti-GFP mAb, is shown in A. (C) Equivalent expression of FcγRs on cells transiently transfected with the indicated constructs was demonstrated by flow cytometry analysis. Surface levels of FcγRs of only transfection-positive cells were assessed as in the legend to Fig. 2. 2.4G2 staining (solid line) and rat IgG control (broken line) are shown. Mock-transfected cells without plasmid DNA were also included as a control. Overexpression of EGFP-PAG3 was confirmed by immunostaining of the mock-transfected cells and cells expressing EGFP-PAG3 with polyclonal anti-PAG3 Abs coupled with Cy3-anti–rabbit IgG, and differences in fluorescent intensities of single cells from these two samples were compared as in the legend to Fig. 2. The level of overexpression of EGFP-PAG3 was estimated to be 23.7 ± 1.1–fold higher than that of endogenous PAG3.

Cooverexpression of ARF6, but not other classes of ARF isoforms, restores the PAG3-induced inhibition of FcγR-mediated phagocytosis. P388D1 cells and cells transiently transfected with the indicated plasmid constructs in pEGFP and pcDNA3 vectors for overexpression were incubated with IgG-opsonized beads, and the phagocytosis index was determined, as described in the legend to Fig. 2. Plasmid DNAs for EGFP-PAG3 (WT) or its GAP-inactive mutant (CA) were cotransfected with plasmid DNAs for ARF1-HA (A), ARF5-HA (B), and ARF6-HA (C). In A–C, 2.0 μg of plasmid DNA for EGFP-PAG3 (bar 2), 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for each ARF-HA (bar 3), and 2.0 μg of plasmid DNA for each ARF-HA (bar 4) were used; the GAP-inactive mutant of EGFP-PAG3 was used instead of wild-type EGFP-PAG3 in bars 5–7, as indicated. In bars 8 and 9 in C, 1.5 μg of plasmid DNA for EGFP-PAG3 plus 0.5 μg of plasmid DNA for the Q67L mutant (bar 8) or the T27N mutant (bar 9) of ARF6-HA were used. Mock-transfected controls were also included (bar 1 in A–C). The overexpression of exogenous proteins were confirmed as described in the legend to Fig. 2 and Fig. 3, with each cell examined for the phagocytic activities. In bar 3 in C, these cells expressed EGFP-PAG3 at levels 25–30-fold higher than endogenous PAG3, and expressed ARF6-HA at levels 7–9-fold higher than endogenous ARF6 (also see Fig. 4 B). In bars 2 and 4 in C, the expression levels of EGFP-PAG3 and ARF6-HA were essentially the same as those described in the legend to Fig. 2 and Fig. 3. Expression of ARF1-HA and ARF5-HA in A and B was also similar to those in Fig. 2. Data are expressed as the mean ± SE of three independent experiments. *P < 0.001.