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Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes.

Cox D, Chang P, Zhang Q, Reddy PG, Bokoch GM, Greenberg S - J. Exp. Med. (1997)

Bottom Line: Specific pathways linking heterotrimeric G proteins and Fcgamma receptors to the actin-based cytoskeleton are poorly understood.Accumulation of F-actin- rich "phagocytic cups" was partially inhibited by expression of Rac1 N17 or Cdc42 N17.In contrast, PMA-induced ruffling was not inhibited by expression of Rac1 N17, but was blocked by expression of Cdc42 N17, indicating that cytoskeletal inhibition by these constructs was nonoverlapping.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Division, Department of Medicine, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
Specific pathways linking heterotrimeric G proteins and Fcgamma receptors to the actin-based cytoskeleton are poorly understood. To test a requirement for Rho family members in cytoskeletal events mediated by structurally diverse receptors in leukocytes, we transfected the full-length human chemotactic peptide receptor in RAW 264.7 cells and examined cytoskeletal alterations in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), colony stimulating factor-1 (CSF-1), IgG-coated particles, and phorbol 12-myristate 13-acetate (PMA). Expression of Rac1 N17, Cdc42 N17, or the GAP domain of n-chimaerin inhibited cytoskeletal responses to FMLP and CSF-1, and blocked phagocytosis. Accumulation of F-actin- rich "phagocytic cups" was partially inhibited by expression of Rac1 N17 or Cdc42 N17. In contrast, PMA-induced ruffling was not inhibited by expression of Rac1 N17, but was blocked by expression of Cdc42 N17, indicating that cytoskeletal inhibition by these constructs was nonoverlapping. These results demonstrate differential requirements for Rho family GTPases in leukocyte motility, and indicate that both Rac1 and Cdc42 are required for Fcgamma receptor- mediated phagocytosis and for membrane ruffling mediated by structurally distinct receptors in macrophages.

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FMLP or CSF-1 induces F-actin–rich ruffles in RAW LacR/ FMLPR.2 cells. Cells were fixed and stained with rhodamine-phalloidin  as described in Materials and Methods. (a) Unstimulated. (b) Cells incubated with 100 nM FMLP for 1 min. (c) Cells incubated with 10 ng/ml  CSF-1 for 5 min. Bar = 10 μm. (d) Inhibition of FMLP-induced ruffles  by pertussis toxin (1 ng/ml for 24 h) in RAW LacR/FMLPR.2 cells.  Solid bars, − pertussis toxin; striped bars, + pertussis toxin.
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Figure 1: FMLP or CSF-1 induces F-actin–rich ruffles in RAW LacR/ FMLPR.2 cells. Cells were fixed and stained with rhodamine-phalloidin as described in Materials and Methods. (a) Unstimulated. (b) Cells incubated with 100 nM FMLP for 1 min. (c) Cells incubated with 10 ng/ml CSF-1 for 5 min. Bar = 10 μm. (d) Inhibition of FMLP-induced ruffles by pertussis toxin (1 ng/ml for 24 h) in RAW LacR/FMLPR.2 cells. Solid bars, − pertussis toxin; striped bars, + pertussis toxin.

Mentions: Although specific macrophage subpopulations respond to the chemotactic peptide, FMLP (19, 20), murine macrophage cell lines lack this responsiveness. To attempt to reconstitute functionally intact FMLP receptors in a murine macrophage cell line, we transfected cDNAs encoding the full-length human FMLP receptor in RAW cells and derived several resultant stable clones. All clones expressed functional FMLP receptors at their surfaces since they bound rhodamine-tagged chemotactic peptide (results not shown). In contrast to quiescent cells, which demonstrated relatively few surface projections (Fig. 1 a), addition of 100 nM FMLP resulted in the appearance of F-actin–rich membrane ruffles in essentially 100% of the transfectants within 15 s of stimulation. No ruffling was seen in untransfected cells incubated with FMLP (results not shown). The ruffling response in the transfectants peaked at 1 min (Fig. 1 b), and had declined by 20 min (not shown). Membrane ruffles appeared diffusely throughout the cell. The extent of ruffling by FMLP, but not by CSF-1, was reduced to control levels in the presence of pertussis toxin (Fig. 1 d), indicating that cytoskeletal coupling by the transfected receptors was mediated by Gi, or a structurally similar heterotrimeric G protein. These data suggest that RAW cells expressing the human FMLP receptor use signal transduction pathways that are similar to those triggered by this serpentine receptor in primary leukocytes.


Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes.

Cox D, Chang P, Zhang Q, Reddy PG, Bokoch GM, Greenberg S - J. Exp. Med. (1997)

FMLP or CSF-1 induces F-actin–rich ruffles in RAW LacR/ FMLPR.2 cells. Cells were fixed and stained with rhodamine-phalloidin  as described in Materials and Methods. (a) Unstimulated. (b) Cells incubated with 100 nM FMLP for 1 min. (c) Cells incubated with 10 ng/ml  CSF-1 for 5 min. Bar = 10 μm. (d) Inhibition of FMLP-induced ruffles  by pertussis toxin (1 ng/ml for 24 h) in RAW LacR/FMLPR.2 cells.  Solid bars, − pertussis toxin; striped bars, + pertussis toxin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199122&req=5

Figure 1: FMLP or CSF-1 induces F-actin–rich ruffles in RAW LacR/ FMLPR.2 cells. Cells were fixed and stained with rhodamine-phalloidin as described in Materials and Methods. (a) Unstimulated. (b) Cells incubated with 100 nM FMLP for 1 min. (c) Cells incubated with 10 ng/ml CSF-1 for 5 min. Bar = 10 μm. (d) Inhibition of FMLP-induced ruffles by pertussis toxin (1 ng/ml for 24 h) in RAW LacR/FMLPR.2 cells. Solid bars, − pertussis toxin; striped bars, + pertussis toxin.
Mentions: Although specific macrophage subpopulations respond to the chemotactic peptide, FMLP (19, 20), murine macrophage cell lines lack this responsiveness. To attempt to reconstitute functionally intact FMLP receptors in a murine macrophage cell line, we transfected cDNAs encoding the full-length human FMLP receptor in RAW cells and derived several resultant stable clones. All clones expressed functional FMLP receptors at their surfaces since they bound rhodamine-tagged chemotactic peptide (results not shown). In contrast to quiescent cells, which demonstrated relatively few surface projections (Fig. 1 a), addition of 100 nM FMLP resulted in the appearance of F-actin–rich membrane ruffles in essentially 100% of the transfectants within 15 s of stimulation. No ruffling was seen in untransfected cells incubated with FMLP (results not shown). The ruffling response in the transfectants peaked at 1 min (Fig. 1 b), and had declined by 20 min (not shown). Membrane ruffles appeared diffusely throughout the cell. The extent of ruffling by FMLP, but not by CSF-1, was reduced to control levels in the presence of pertussis toxin (Fig. 1 d), indicating that cytoskeletal coupling by the transfected receptors was mediated by Gi, or a structurally similar heterotrimeric G protein. These data suggest that RAW cells expressing the human FMLP receptor use signal transduction pathways that are similar to those triggered by this serpentine receptor in primary leukocytes.

Bottom Line: Specific pathways linking heterotrimeric G proteins and Fcgamma receptors to the actin-based cytoskeleton are poorly understood.Accumulation of F-actin- rich "phagocytic cups" was partially inhibited by expression of Rac1 N17 or Cdc42 N17.In contrast, PMA-induced ruffling was not inhibited by expression of Rac1 N17, but was blocked by expression of Cdc42 N17, indicating that cytoskeletal inhibition by these constructs was nonoverlapping.

View Article: PubMed Central - PubMed

Affiliation: Pulmonary Division, Department of Medicine, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
Specific pathways linking heterotrimeric G proteins and Fcgamma receptors to the actin-based cytoskeleton are poorly understood. To test a requirement for Rho family members in cytoskeletal events mediated by structurally diverse receptors in leukocytes, we transfected the full-length human chemotactic peptide receptor in RAW 264.7 cells and examined cytoskeletal alterations in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), colony stimulating factor-1 (CSF-1), IgG-coated particles, and phorbol 12-myristate 13-acetate (PMA). Expression of Rac1 N17, Cdc42 N17, or the GAP domain of n-chimaerin inhibited cytoskeletal responses to FMLP and CSF-1, and blocked phagocytosis. Accumulation of F-actin- rich "phagocytic cups" was partially inhibited by expression of Rac1 N17 or Cdc42 N17. In contrast, PMA-induced ruffling was not inhibited by expression of Rac1 N17, but was blocked by expression of Cdc42 N17, indicating that cytoskeletal inhibition by these constructs was nonoverlapping. These results demonstrate differential requirements for Rho family GTPases in leukocyte motility, and indicate that both Rac1 and Cdc42 are required for Fcgamma receptor- mediated phagocytosis and for membrane ruffling mediated by structurally distinct receptors in macrophages.

Show MeSH
Related in: MedlinePlus