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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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Inhibition of FcγR-mediated phagocytosis by Cdc42 N17.  Phagocytosis assay was performed as described in Materials and Methods.  (a) Phase-contrast micrograph. (b) Fluorescence micrograph of rhodamine  anti–rabbit IgG-stained cells to indicate presence of IgG-RBCs. After fixation and permeabilization, uningested erythrocytes appear crenated. (c)  Fluorescence micrograph of anti-Myc–stained cell to indicate expression  of Myc-tagged Cdc42 N17. Note ingestion of IgG-RBCs in neighboring  cells not expressing the Myc epitope.
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Figure 5: Inhibition of FcγR-mediated phagocytosis by Cdc42 N17. Phagocytosis assay was performed as described in Materials and Methods. (a) Phase-contrast micrograph. (b) Fluorescence micrograph of rhodamine anti–rabbit IgG-stained cells to indicate presence of IgG-RBCs. After fixation and permeabilization, uningested erythrocytes appear crenated. (c) Fluorescence micrograph of anti-Myc–stained cell to indicate expression of Myc-tagged Cdc42 N17. Note ingestion of IgG-RBCs in neighboring cells not expressing the Myc epitope.

Mentions: FcγR-mediated phagocytosis requires the net assembly of actin which forms the structural scaffolding that constitutes phagocytic cups. To determine whether Rac1 or Cdc42 were required for phagocytosis, we performed phagocytosis assays on cell populations induced with IPTG and compared ingestion of IgG-RBCs in cells that either did or did not express the various fusion proteins. For example, expression of Cdc42 N17 blocked ingestion of IgG-RBCs, despite the presence of surface-bound erythrocytes (Fig. 5). Expression of Rac1 N17, Cdc42 N17, or Chimaerin-GAP profoundly inhibited phagocytosis (Fig. 6). In contrast, expression of the fusion constructs had a modest effect on particle binding. For example, expression of Rac1 N17 led to a 27 ± 2.7% reduction in particle binding, whereas expression of Cdc42 N17 reduced binding by 38 ± 9.7%. In contrast, Chimaerin-GAP had no effect on binding efficiency (i.e., the average number of IgG-RBCs associated with either induced or uninduced cells was seven). Thus, inhibition of Rac1 and Cdc42 function by either dominant-negative versions of these proteins or by GAP expression led to a disproportionate decrease in phagocytosis, as compared to binding, of IgG-RBCs.


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)

Inhibition of FcγR-mediated phagocytosis by Cdc42 N17.  Phagocytosis assay was performed as described in Materials and Methods.  (a) Phase-contrast micrograph. (b) Fluorescence micrograph of rhodamine  anti–rabbit IgG-stained cells to indicate presence of IgG-RBCs. After fixation and permeabilization, uningested erythrocytes appear crenated. (c)  Fluorescence micrograph of anti-Myc–stained cell to indicate expression  of Myc-tagged Cdc42 N17. Note ingestion of IgG-RBCs in neighboring  cells not expressing the Myc epitope.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Inhibition of FcγR-mediated phagocytosis by Cdc42 N17. Phagocytosis assay was performed as described in Materials and Methods. (a) Phase-contrast micrograph. (b) Fluorescence micrograph of rhodamine anti–rabbit IgG-stained cells to indicate presence of IgG-RBCs. After fixation and permeabilization, uningested erythrocytes appear crenated. (c) Fluorescence micrograph of anti-Myc–stained cell to indicate expression of Myc-tagged Cdc42 N17. Note ingestion of IgG-RBCs in neighboring cells not expressing the Myc epitope.
Mentions: FcγR-mediated phagocytosis requires the net assembly of actin which forms the structural scaffolding that constitutes phagocytic cups. To determine whether Rac1 or Cdc42 were required for phagocytosis, we performed phagocytosis assays on cell populations induced with IPTG and compared ingestion of IgG-RBCs in cells that either did or did not express the various fusion proteins. For example, expression of Cdc42 N17 blocked ingestion of IgG-RBCs, despite the presence of surface-bound erythrocytes (Fig. 5). Expression of Rac1 N17, Cdc42 N17, or Chimaerin-GAP profoundly inhibited phagocytosis (Fig. 6). In contrast, expression of the fusion constructs had a modest effect on particle binding. For example, expression of Rac1 N17 led to a 27 ± 2.7% reduction in particle binding, whereas expression of Cdc42 N17 reduced binding by 38 ± 9.7%. In contrast, Chimaerin-GAP had no effect on binding efficiency (i.e., the average number of IgG-RBCs associated with either induced or uninduced cells was seven). Thus, inhibition of Rac1 and Cdc42 function by either dominant-negative versions of these proteins or by GAP expression led to a disproportionate decrease in phagocytosis, as compared to binding, of IgG-RBCs.

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