ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting - PubMed (original) (raw)

ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting

Q Zhang et al. Mol Cell Biol. 1999 Dec.

Abstract

Activation of Rac1, a member of the Rho family of GTPases, is associated with multiple cellular responses, including membrane ruffling and focal complex formation. The mechanisms by which Rac1 is coupled to these functional responses are not well understood. It was recently shown that ARF6, a GTPase implicated in cytoskeletal alterations and a membrane recycling pathway, is required for Rac1-dependent phagocytosis in macrophages (Q. Zhang et al., J. Biol. Chem. 273:19977-19981, 1998). To determine whether ARF6 is required for Rac1-dependent cytoskeletal responses in macrophages, we expressed wild-type (WT) or guanine nucleotide binding-deficient alleles (T27N) of ARF6 in macrophages coexpressing activated alleles of Rac1 (Q61L) or Cdc42 (Q61L) or stimulated with colony-stimulating factor 1 (CSF-1). Expression of ARF6 T27N but not ARF6 WT inhibited ruffles mediated by Rac1 Q61L or CSF-1. In contrast, expression of ARF6 T27N did not inhibit Rac1 Q61L-mediated focal complex formation and did not impair Cdc42 Q61L-mediated filopodial formation. Cryoimmunogold electron microscopy demonstrated the presence of ARF6 in membrane ruffles induced by either CSF-1 or Rac1 Q61L. Addition of CSF-1 to macrophages led to the redistribution of ARF6 from the interior of the cell to the plasma membrane, suggesting that this growth factor triggers ARF6 activation. Direct targeting of Rac1 to the plasma membrane did not bypass the blockade in ruffling induced by ARF6 T27N, indicating that ARF6 regulates a pathway leading to membrane ruffling that occurs after the activation and membrane association of Rac. These data demonstrate that intact ARF6 function is required for coupling activated Rac to one of several effector pathways and suggest that a principal function of ARF6 is to coordinate Rac activation with plasma membrane-based protrusive events.

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Figures

FIG. 1

FIG. 1

Effect of ARF6 T27N on Rac1- and Cdc42-mediated cytoskeletal alterations in macrophages. Adherent RAW LacR/FMLPR.2 cells transfected with Myc-Rac1 Q61L (Rac), Myc-Cdc42 Q61L (Cdc42), ARF6 WT, or ARF6 T27N were fixed and stained with a MAb against Myc (to detect either Rac or Cdc42), rabbit antiserum against ARF6, and rhodamine-phalloidin (to detect F-actin). Confocal fluorescence micrographs showing rhodamine-phalloidin fluorescence micrographs of representative cells are shown. Bar = 10 μm.

FIG. 2

FIG. 2

Quantitation of membrane ruffling or filopodia formation of Rac1 Q61L (A)- or Cdc42 61L (B)-expressing macrophages also expressing the indicated ARF6 alleles. Data represent the means ± SEM (n = 4).

FIG. 3

FIG. 3

Effect of ARF6 T27N on focal complex formation induced by Rac1 Q61L in macrophages. Adherent RAW LacR/FMLPR.2 cells transfected with the indicated constructs were fixed and stained with a MAb against Myc (to detect Myc-Rac1 Q61L), rabbit antiserum against ARF6 (to detect cells expressing ARF6 T27N), and MAb PY-99 (to detect phosphotyrosine). Representative confocal micrographs showing Myc (left panels) and PY-99 staining (right panels) are shown. Note prominent phosphotyrosine-rich focal complexes in cells expressing Myc-Rac1 Q61L but not in surrounding nonexpressors. Bar = 10 μm.

FIG. 4

FIG. 4

Effect of ARF T27N on CSF-1-induced membrane ruffling in macrophages. Adherent RAW LacR/FMLPR.2 cells transfected with the indicated constructs were incubated in the presence or absence of 10 ng of CSF-1 per ml for 5 min at 37°C, fixed, and stained with anti-ARF6 and rhodamine-phalloidin. Representative confocal fluorescence micrographs are shown. Bar = 10 μm.

FIG. 5

FIG. 5

Quantitation of membrane ruffling induced by CSF-1 in macrophages expressing the indicated constructs in nonexpressing controls. Data represent the means ± SEM (n = 5).

FIG. 6

FIG. 6

Effect of CSF-1 on the localization of ARF6 in macrophages. Adherent RAW LacR/FMLPR.2 cells transfected with HA-ARF6 were incubated in the absence (A) or presence (B) of 10 ng of CSF-1 for 5 min at 37°C. Ultrathin cryosections were labeled with monoclonal anti-HA followed by rabbit anti-mouse IgG and anti-rabbit IgG–10-nm-diameter gold. ARF6 localized to the plasma membrane (p) and intracellular vesicles and scattered throughout the cytosol. There is also some labeling in the mitochondria (m). Membrane ruffles (r) are apparent in cells incubated with CSF-1 (B). Note that the density of ARF6 labeling is the same for the ruffles and the adjacent areas of the plasma membrane. n, nucleus. Bars = 500 nm.

FIG. 7

FIG. 7

Localization of ARF6 and Rac1 in cells coexpressing Myc-Rac1 Q61L and HA-ARF6 WT. Cryosections of adherent RAW LacR/FMLPR.2 cells transfected with the indicated constructs were double labeled with mouse anti-HA followed by rabbit anti-mouse IgG and anti-rabbit IgG–5-nm-diameter gold and with mouse anti-Myc followed by anti-mouse IgG–10-nm-diameter gold. Labeling with Rac1 (10-nm-diameter gold) can be seen on the plasma membrane (p) and on the membrane of the ruffles (r). The insets show a higher magnification of the marked areas showing the labeling with ARF6 (5-nm-diameter gold) and Rac1 (10-nm-diameter gold). The intensity of both labels on the membrane ruffles and on adjacent nonruffling areas of the plasma membrane is equivalent. Bars = 300 nm.

FIG. 8

FIG. 8

Localization of HA-ARF6 T27N in cells coexpressing Myc-Rac1 Q61L. Cryosections of adherent RAW LacR/FMLPR.2 cells transfected with the indicated constructs were double labeled with a mixture of rabbit anti-HA and mouse anti-Myc followed by a mixture of goat anti-rabbit IgG–10-nm-diameter gold and goat anti-mouse IgG–5-nm-diameter gold. (A) Low magnification of a cell expressing both constructs. n, nucleus. (B) Higher magnification of marked area in panel A showing the plasma membrane labeled only with Rac1 (5-nm-diameter gold) (arrows). (C) Higher magnification of the marked area in panel A showing endocytic vesicles (e) labeled for both ARF6 (10-nm-diameter gold) (large arrows) and Rac1 (5-nm-diameter gold) (small arrows). m, mitochondria. Bars = 1,000 nm (A), 100 nm (B), and 200 nm (C).

FIG. 9

FIG. 9

Effect of ARF6 T27N on membrane ruffles induced by expression of a plasma membrane-targeted fusion protein containing Rac1 Q61L in macrophages. RAW LacR/FMLPR.2 cells were transfected with either 16:7:Myc-Rac1 Q61L or 16:7:Myc-Rac1 Q61L and ARF6 T27N. Expression of 16:7:Myc-Rac1 Q61L at the plasma membrane was confirmed by cell surface staining of CD16 (left panels); F-actin was visualized following subsequent permeabilization and staining with rhodamine-phalloidin (right panels). Bar = 10 μm.

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