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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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Ruffling indices of  PMA-stimulated RAW cells  transfected with the indicated constructs. Striped bars, Myc-expressing cells; solid bars, controls, which  are derived from the same clones  but did not express the Myc  epitope by indirect immunofluorescence. Ruffling indices of cells  incubated in the absence of  IPTG, zinc, and butyrate were  indistinguishable from cells that  were incubated with these agents  but did not express Myc. Data are expressed as the mean ± SEM, n = 3.  The reduction of ruffling by Cdc42 N17 or Chimaerin-GAP was statistically significant (P <0.005).
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Figure 9: Ruffling indices of PMA-stimulated RAW cells transfected with the indicated constructs. Striped bars, Myc-expressing cells; solid bars, controls, which are derived from the same clones but did not express the Myc epitope by indirect immunofluorescence. Ruffling indices of cells incubated in the absence of IPTG, zinc, and butyrate were indistinguishable from cells that were incubated with these agents but did not express Myc. Data are expressed as the mean ± SEM, n = 3. The reduction of ruffling by Cdc42 N17 or Chimaerin-GAP was statistically significant (P <0.005).

Mentions: The qualitatively similar effects of Cdc42 N17 and Rac1 N17 on responses mediated by structurally distinct ligands (i.e., FMLP, CSF-1, and IgG) raised the possibility that expression of Rac1 N17 or Cdc42 N17 led to nonspecific toxic effects on RAW cells, rendering them incapable of responding to any F-actin–mobilizing agonist. This was of particular concern in cells expressing high levels of Rac1 N17, which caused cell rounding. We tested several other ligands for their ability to induce cytoskeletal changes in these cells, but could not detect morphological changes after addition of lysophosphatidic acid, bradykinin, platelet activating factor, or ATP (results not shown). However, addition of PMA caused dramatic changes in cell shape, leading to cell spreading and membrane ruffling (Fig. 8), both of which were inhibited by 1 μM cytochalasin D (results not shown). In marked contrast to the above results using FMLP, CSF-1, or IgG-RBCs, expression of Rac1 N17 did not inhibit PMA-induced ruffling (compare Fig. 8 b with c). However, expression of Cdc42 N17 blocked PMA-induced ruffling (Fig. 8 d and Fig. 9). These results indicate that expression of Rac1 N17 did not lead to a global lack of responsiveness in these cells. PMA-induced cellular responses are often attributed to activation of one or more protein kinase C isoforms. We could not confirm or refute this, since addition of 5 μM calphostin C inhibited PMA-induced membrane ruffling, whereas other inhibitors of protein kinase C, such as chelerythrine chloride (10 μM) and staurosporine (1 μM), did not (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)

Ruffling indices of  PMA-stimulated RAW cells  transfected with the indicated constructs. Striped bars, Myc-expressing cells; solid bars, controls, which  are derived from the same clones  but did not express the Myc  epitope by indirect immunofluorescence. Ruffling indices of cells  incubated in the absence of  IPTG, zinc, and butyrate were  indistinguishable from cells that  were incubated with these agents  but did not express Myc. Data are expressed as the mean ± SEM, n = 3.  The reduction of ruffling by Cdc42 N17 or Chimaerin-GAP was statistically significant (P <0.005).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199122&req=5

Figure 9: Ruffling indices of PMA-stimulated RAW cells transfected with the indicated constructs. Striped bars, Myc-expressing cells; solid bars, controls, which are derived from the same clones but did not express the Myc epitope by indirect immunofluorescence. Ruffling indices of cells incubated in the absence of IPTG, zinc, and butyrate were indistinguishable from cells that were incubated with these agents but did not express Myc. Data are expressed as the mean ± SEM, n = 3. The reduction of ruffling by Cdc42 N17 or Chimaerin-GAP was statistically significant (P <0.005).
Mentions: The qualitatively similar effects of Cdc42 N17 and Rac1 N17 on responses mediated by structurally distinct ligands (i.e., FMLP, CSF-1, and IgG) raised the possibility that expression of Rac1 N17 or Cdc42 N17 led to nonspecific toxic effects on RAW cells, rendering them incapable of responding to any F-actin–mobilizing agonist. This was of particular concern in cells expressing high levels of Rac1 N17, which caused cell rounding. We tested several other ligands for their ability to induce cytoskeletal changes in these cells, but could not detect morphological changes after addition of lysophosphatidic acid, bradykinin, platelet activating factor, or ATP (results not shown). However, addition of PMA caused dramatic changes in cell shape, leading to cell spreading and membrane ruffling (Fig. 8), both of which were inhibited by 1 μM cytochalasin D (results not shown). In marked contrast to the above results using FMLP, CSF-1, or IgG-RBCs, expression of Rac1 N17 did not inhibit PMA-induced ruffling (compare Fig. 8 b with c). However, expression of Cdc42 N17 blocked PMA-induced ruffling (Fig. 8 d and Fig. 9). These results indicate that expression of Rac1 N17 did not lead to a global lack of responsiveness in these cells. PMA-induced cellular responses are often attributed to activation of one or more protein kinase C isoforms. We could not confirm or refute this, since addition of 5 μM calphostin C inhibited PMA-induced membrane ruffling, whereas other inhibitors of protein kinase C, such as chelerythrine chloride (10 μM) and staurosporine (1 μM), did not (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