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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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Phagocytic cup formation is inhibited by expression of Rac1  N17 or Cdc42 N17. Cells were incubated with IgG-RBCs for 5 min at  37°C before fixation. Immunofluorescence using rhodamine-phalloidin and  anti–rabbit IgG was performed as described in Materials and Methods. a,  c, and e, rhodamine-phalloidin; b, d, and f, anti–rabbit IgG. a and b: control; c and d, Cdc42 N17; e and f, Rac1 N17. Bar = 10 μm.
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Figure 7: Phagocytic cup formation is inhibited by expression of Rac1 N17 or Cdc42 N17. Cells were incubated with IgG-RBCs for 5 min at 37°C before fixation. Immunofluorescence using rhodamine-phalloidin and anti–rabbit IgG was performed as described in Materials and Methods. a, c, and e, rhodamine-phalloidin; b, d, and f, anti–rabbit IgG. a and b: control; c and d, Cdc42 N17; e and f, Rac1 N17. Bar = 10 μm.

Mentions: To determine whether the inhibition of phagocytosis correlated with an inhibition of the submembranous accumulations of F-actin, we fixed and stained various clones of RAW cells undergoing early stages of phagocytosis. 5 min after the onset of phagocytosis, F-actin–rich phagocytic cups were clearly visible in control cells (Fig. 7, a and b), confirming earlier data (15). Expression of Rac1 N17 or Cdc42 N17 (Fig. 7, c–f  ) inhibited the appearance of distinct F-actin– rich cups, although some focal accumulations of F-actin beneath the test particles did occur in these cells. The inhibition of phagocytic cup formation by Cdc42 N17 was usually incomplete, whereas expression of Rac1 N17 abolished much of the focal appearance of F-actin beneath the test particles (compare Fig. 7 e with c). The effects of Chimaerin-GAP resembled that of Cdc42 N17 (results not shown).


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)

Phagocytic cup formation is inhibited by expression of Rac1  N17 or Cdc42 N17. Cells were incubated with IgG-RBCs for 5 min at  37°C before fixation. Immunofluorescence using rhodamine-phalloidin and  anti–rabbit IgG was performed as described in Materials and Methods. a,  c, and e, rhodamine-phalloidin; b, d, and f, anti–rabbit IgG. a and b: control; c and d, Cdc42 N17; e and f, Rac1 N17. Bar = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199122&req=5

Figure 7: Phagocytic cup formation is inhibited by expression of Rac1 N17 or Cdc42 N17. Cells were incubated with IgG-RBCs for 5 min at 37°C before fixation. Immunofluorescence using rhodamine-phalloidin and anti–rabbit IgG was performed as described in Materials and Methods. a, c, and e, rhodamine-phalloidin; b, d, and f, anti–rabbit IgG. a and b: control; c and d, Cdc42 N17; e and f, Rac1 N17. Bar = 10 μm.
Mentions: To determine whether the inhibition of phagocytosis correlated with an inhibition of the submembranous accumulations of F-actin, we fixed and stained various clones of RAW cells undergoing early stages of phagocytosis. 5 min after the onset of phagocytosis, F-actin–rich phagocytic cups were clearly visible in control cells (Fig. 7, a and b), confirming earlier data (15). Expression of Rac1 N17 or Cdc42 N17 (Fig. 7, c–f  ) inhibited the appearance of distinct F-actin– rich cups, although some focal accumulations of F-actin beneath the test particles did occur in these cells. The inhibition of phagocytic cup formation by Cdc42 N17 was usually incomplete, whereas expression of Rac1 N17 abolished much of the focal appearance of F-actin beneath the test particles (compare Fig. 7 e with c). The effects of Chimaerin-GAP resembled that of Cdc42 N17 (results not shown).

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