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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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Expression of Myc-tagged Rac1 N17, Cdc42 N17, or Chimaerin-GAP inhibits ruffling in response to FMLP. Cells were incubated  with 100 nM FMLP for 1 min before fixation and staining with rhodamine-phalloidin to detect F-actin and mAb anti-Myc followed by  FITC anti–mouse IgG to detect the indicated fusion proteins, as described  in Materials and Methods. Rhodamine-phalloidin staining of a control  cell is shown in a and of representative Myc-positive cells are shown in b–d.  A field of cells, some of which express Chimaerin-GAP, is depicted in e  (stained with anti-Myc) and f (stained with rhodamine-phalloidin). a, Control; b, Rac1 N17; c, Cdc42 N17; d–f, Chimaerin-GAP. Bar = 10 μm.
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Figure 3: Expression of Myc-tagged Rac1 N17, Cdc42 N17, or Chimaerin-GAP inhibits ruffling in response to FMLP. Cells were incubated with 100 nM FMLP for 1 min before fixation and staining with rhodamine-phalloidin to detect F-actin and mAb anti-Myc followed by FITC anti–mouse IgG to detect the indicated fusion proteins, as described in Materials and Methods. Rhodamine-phalloidin staining of a control cell is shown in a and of representative Myc-positive cells are shown in b–d. A field of cells, some of which express Chimaerin-GAP, is depicted in e (stained with anti-Myc) and f (stained with rhodamine-phalloidin). a, Control; b, Rac1 N17; c, Cdc42 N17; d–f, Chimaerin-GAP. Bar = 10 μm.

Mentions: The morphology of cells expressing the various Myc-tagged fusion constructs was dependent on the specific construct expressed and its level of expression. Relatively high levels of expression of Rac1 N17, obtainable by the addition of zinc and butyrate, were associated with cell rounding (for example, see Fig. 3 b), whereas moderate expression of either construct did not lead to an altered cell morphology (results not shown). Expression of Cdc42 N17 did not significantly affect the morphology of the cells (Fig. 3 c). Cells expressing Chimaerin-GAP frequently appeared more spread than control cells (Fig. 3 d).


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)

Expression of Myc-tagged Rac1 N17, Cdc42 N17, or Chimaerin-GAP inhibits ruffling in response to FMLP. Cells were incubated  with 100 nM FMLP for 1 min before fixation and staining with rhodamine-phalloidin to detect F-actin and mAb anti-Myc followed by  FITC anti–mouse IgG to detect the indicated fusion proteins, as described  in Materials and Methods. Rhodamine-phalloidin staining of a control  cell is shown in a and of representative Myc-positive cells are shown in b–d.  A field of cells, some of which express Chimaerin-GAP, is depicted in e  (stained with anti-Myc) and f (stained with rhodamine-phalloidin). a, Control; b, Rac1 N17; c, Cdc42 N17; d–f, Chimaerin-GAP. Bar = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Expression of Myc-tagged Rac1 N17, Cdc42 N17, or Chimaerin-GAP inhibits ruffling in response to FMLP. Cells were incubated with 100 nM FMLP for 1 min before fixation and staining with rhodamine-phalloidin to detect F-actin and mAb anti-Myc followed by FITC anti–mouse IgG to detect the indicated fusion proteins, as described in Materials and Methods. Rhodamine-phalloidin staining of a control cell is shown in a and of representative Myc-positive cells are shown in b–d. A field of cells, some of which express Chimaerin-GAP, is depicted in e (stained with anti-Myc) and f (stained with rhodamine-phalloidin). a, Control; b, Rac1 N17; c, Cdc42 N17; d–f, Chimaerin-GAP. Bar = 10 μm.
Mentions: The morphology of cells expressing the various Myc-tagged fusion constructs was dependent on the specific construct expressed and its level of expression. Relatively high levels of expression of Rac1 N17, obtainable by the addition of zinc and butyrate, were associated with cell rounding (for example, see Fig. 3 b), whereas moderate expression of either construct did not lead to an altered cell morphology (results not shown). Expression of Cdc42 N17 did not significantly affect the morphology of the cells (Fig. 3 c). Cells expressing Chimaerin-GAP frequently appeared more spread than control cells (Fig. 3 d).

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