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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 and scores of FMLP- or CSF-1–stimulated  RAW cells transfected with the indicated constructs. Narrow striped bars,  Myc-expressing cells; solid bars, controls, derived from the same clones but  which 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. a, FMLP; b, CSF-1. Data are expressed as  the mean ± SEM, n = 3. The reduction of ruffling by Rac1 N17, Cdc42  N17, or Chimaerin-GAP was statistically significant (P <0.005). (c) Histograms of ruffling scores of individual cells stimulated with the indicated  agonist. The extent of ruffling of each cell was scored using a scale of 0–2,  where 0 indicates that no ruffles were present, 1 indicates that ruffling was  confined to one area of the cell only (<25% of cell circumference), and 2  indicates that two or more discrete areas of the cell contained ruffles. 140  cells were counted for each construct.
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Figure 4: Ruffling indices and scores of FMLP- or CSF-1–stimulated RAW cells transfected with the indicated constructs. Narrow striped bars, Myc-expressing cells; solid bars, controls, derived from the same clones but which 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. a, FMLP; b, CSF-1. Data are expressed as the mean ± SEM, n = 3. The reduction of ruffling by Rac1 N17, Cdc42 N17, or Chimaerin-GAP was statistically significant (P <0.005). (c) Histograms of ruffling scores of individual cells stimulated with the indicated agonist. The extent of ruffling of each cell was scored using a scale of 0–2, where 0 indicates that no ruffles were present, 1 indicates that ruffling was confined to one area of the cell only (<25% of cell circumference), and 2 indicates that two or more discrete areas of the cell contained ruffles. 140 cells were counted for each construct.

Mentions: Similar results were obtained using CSF-1 as a ruffle-inducing agent. Expression of Rac1 N17, Cdc42 N17, or Chimaerin-GAP inhibited CSF-induced membrane ruffling by 88–96%. The inhibitory effects on membrane ruffling of Rac1 N17 and Cdc42 N17 were apparent in terms of the number of cells that demonstrated no ruffling response and in the extent of ruffling in those cells that did respond to either FMLP or CSF-1 (Fig. 4 c).


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 and scores of FMLP- or CSF-1–stimulated  RAW cells transfected with the indicated constructs. Narrow striped bars,  Myc-expressing cells; solid bars, controls, derived from the same clones but  which 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. a, FMLP; b, CSF-1. Data are expressed as  the mean ± SEM, n = 3. The reduction of ruffling by Rac1 N17, Cdc42  N17, or Chimaerin-GAP was statistically significant (P <0.005). (c) Histograms of ruffling scores of individual cells stimulated with the indicated  agonist. The extent of ruffling of each cell was scored using a scale of 0–2,  where 0 indicates that no ruffles were present, 1 indicates that ruffling was  confined to one area of the cell only (<25% of cell circumference), and 2  indicates that two or more discrete areas of the cell contained ruffles. 140  cells were counted for each construct.
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Related In: Results  -  Collection

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Figure 4: Ruffling indices and scores of FMLP- or CSF-1–stimulated RAW cells transfected with the indicated constructs. Narrow striped bars, Myc-expressing cells; solid bars, controls, derived from the same clones but which 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. a, FMLP; b, CSF-1. Data are expressed as the mean ± SEM, n = 3. The reduction of ruffling by Rac1 N17, Cdc42 N17, or Chimaerin-GAP was statistically significant (P <0.005). (c) Histograms of ruffling scores of individual cells stimulated with the indicated agonist. The extent of ruffling of each cell was scored using a scale of 0–2, where 0 indicates that no ruffles were present, 1 indicates that ruffling was confined to one area of the cell only (<25% of cell circumference), and 2 indicates that two or more discrete areas of the cell contained ruffles. 140 cells were counted for each construct.
Mentions: Similar results were obtained using CSF-1 as a ruffle-inducing agent. Expression of Rac1 N17, Cdc42 N17, or Chimaerin-GAP inhibited CSF-induced membrane ruffling by 88–96%. The inhibitory effects on membrane ruffling of Rac1 N17 and Cdc42 N17 were apparent in terms of the number of cells that demonstrated no ruffling response and in the extent of ruffling in those cells that did respond to either FMLP or CSF-1 (Fig. 4 c).

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