Limits...
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.

Show MeSH

Related in: MedlinePlus

Induction of expression of Myc-tagged fusion proteins in stable RAW cell transfectants. (a) 106 RAW cells  (derived from a clone of RAW  LacR/FMLPR.2 cells transfected  with Myc-tagged Chimaerin-GAP) were incubated for 0, 5, or  15 h in the presence or absence of  10 mM IPTG, 50 μM zinc, and  2 mM butyrate. Cells were subjected to detergent lysis, SDS-PAGE, and immunoblotting  with either anti-Myc or antiactin  mAbs. Lanes 1 and 4, uninduced; lanes 2 and 3, induced  with IPTG only; lanes 5 and 6,  induced with IPTG, butyrate,  and zinc. (b) Average levels of  expression of Myc-tagged fusion  proteins in individual cells. Induction of protein expression  was performed as described in  Materials and Methods and cells were fixed and stained with anti-Myc  mAb and processed for microspectrofluorometry. Expression levels are  shown in arbitrary fluorescence units. Nonspecific fluorescence (i.e., of  uninduced cells or untransfected cells) was <10% of the total fluorescence signal. Data are expressed as the mean fluorescence (± SEM) of  150–200 Myc-positive cells and represent all Myc-positive cells observed  in 5–10 high power fields from three separate experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199122&req=5

Figure 2: Induction of expression of Myc-tagged fusion proteins in stable RAW cell transfectants. (a) 106 RAW cells (derived from a clone of RAW LacR/FMLPR.2 cells transfected with Myc-tagged Chimaerin-GAP) were incubated for 0, 5, or 15 h in the presence or absence of 10 mM IPTG, 50 μM zinc, and 2 mM butyrate. Cells were subjected to detergent lysis, SDS-PAGE, and immunoblotting with either anti-Myc or antiactin mAbs. Lanes 1 and 4, uninduced; lanes 2 and 3, induced with IPTG only; lanes 5 and 6, induced with IPTG, butyrate, and zinc. (b) Average levels of expression of Myc-tagged fusion proteins in individual cells. Induction of protein expression was performed as described in Materials and Methods and cells were fixed and stained with anti-Myc mAb and processed for microspectrofluorometry. Expression levels are shown in arbitrary fluorescence units. Nonspecific fluorescence (i.e., of uninduced cells or untransfected cells) was <10% of the total fluorescence signal. Data are expressed as the mean fluorescence (± SEM) of 150–200 Myc-positive cells and represent all Myc-positive cells observed in 5–10 high power fields from three separate experiments.

Mentions: To test a requirement for Rac1 and Cdc42 on membrane ruffling in RAW cells, we expressed inducible GTP binding–deficient versions of these proteins in RAW cells and selected stable clones. As a complementary approach, we also utilized the GAP domain of n-chimaerin (Chimaerin-GAP), previously shown to display GAP activity towards Rac1 and Cdc42 (22). Expression of Chimaerin-GAP would be expected to decrease the accumulation of GTP-bound (active) forms of Rac1 and Cdc42. We used a RAW cell clone (RAW LacR/FMLPR.2) that stably expressed both the human FMLP receptor and the Lac repressor protein as a recipient cell line. This line was used to generate further clones expressing Myc-tagged human Rac1 N17, Cdc42 N17, and Chimaerin-GAP. Between 50 and 200 clones expressing each construct were isolated. The clones varied in their “leakiness” (i.e., expression of the Myc epitope in the absence of the inducing agent, IPTG) and level of inducible expression. Therefore, we determined optimal conditions for the expression of the constructs, and selected three to six clones that displayed a high level of inducible expression for each construct. For example, in the absence of IPTG, minimal expression of the Myc epitope was detected, whereas IPTG caused a time-dependent appearance of fusion protein expression. The addition of zinc acetate and sodium butyrate further enhanced protein expression (Fig. 2 a). Fluorescence microscopy revealed a bimodal population of cells that either did or did not express the Myc epitope. In uninduced cells, <1% of the cells expressed the Myc epitope, whereas addition of IPTG, zinc, and butyrate induced expression of the Myc epitope in 20–60% of the total cell population. Clones were selected on the basis of demonstrating uniformly bright fluorescence in those individual cells that stained positive for the Myc epitope (i.e., cells within a selected clone showed a sharp contrast between Myc expression and a lack of expression, and the variation of intensities of Myc-positive cells was relatively small). We quantitated expression of the Myc-tagged fusion proteins on a single cell basis by measuring the fluorescence intensities of Myc-positive cells using microspectrofluorometry. Clones that inducibly expressed Rac1 N17 or Cdc42 N17 showed similar levels of expression, whereas Chimaerin-GAP consistently showed a greater level of expression on a single cell basis (P <0.05, when compared to any other clone; Fig. 2 b). In the 40–80% of the population that did not stain positive with mAb anti-Myc despite the presence of inducing agents, the mean fluorescence was similar to that of uninduced cells. Although we cannot explain why only a proportion of cells of a given clone expressed the fusion proteins, the nonexpressors of a given clone provided ideal controls to test the effects of fusion protein expression on cytoskeletal responses.


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)

Induction of expression of Myc-tagged fusion proteins in stable RAW cell transfectants. (a) 106 RAW cells  (derived from a clone of RAW  LacR/FMLPR.2 cells transfected  with Myc-tagged Chimaerin-GAP) were incubated for 0, 5, or  15 h in the presence or absence of  10 mM IPTG, 50 μM zinc, and  2 mM butyrate. Cells were subjected to detergent lysis, SDS-PAGE, and immunoblotting  with either anti-Myc or antiactin  mAbs. Lanes 1 and 4, uninduced; lanes 2 and 3, induced  with IPTG only; lanes 5 and 6,  induced with IPTG, butyrate,  and zinc. (b) Average levels of  expression of Myc-tagged fusion  proteins in individual cells. Induction of protein expression  was performed as described in  Materials and Methods and cells were fixed and stained with anti-Myc  mAb and processed for microspectrofluorometry. Expression levels are  shown in arbitrary fluorescence units. Nonspecific fluorescence (i.e., of  uninduced cells or untransfected cells) was <10% of the total fluorescence signal. Data are expressed as the mean fluorescence (± SEM) of  150–200 Myc-positive cells and represent all Myc-positive cells observed  in 5–10 high power fields from three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Induction of expression of Myc-tagged fusion proteins in stable RAW cell transfectants. (a) 106 RAW cells (derived from a clone of RAW LacR/FMLPR.2 cells transfected with Myc-tagged Chimaerin-GAP) were incubated for 0, 5, or 15 h in the presence or absence of 10 mM IPTG, 50 μM zinc, and 2 mM butyrate. Cells were subjected to detergent lysis, SDS-PAGE, and immunoblotting with either anti-Myc or antiactin mAbs. Lanes 1 and 4, uninduced; lanes 2 and 3, induced with IPTG only; lanes 5 and 6, induced with IPTG, butyrate, and zinc. (b) Average levels of expression of Myc-tagged fusion proteins in individual cells. Induction of protein expression was performed as described in Materials and Methods and cells were fixed and stained with anti-Myc mAb and processed for microspectrofluorometry. Expression levels are shown in arbitrary fluorescence units. Nonspecific fluorescence (i.e., of uninduced cells or untransfected cells) was <10% of the total fluorescence signal. Data are expressed as the mean fluorescence (± SEM) of 150–200 Myc-positive cells and represent all Myc-positive cells observed in 5–10 high power fields from three separate experiments.
Mentions: To test a requirement for Rac1 and Cdc42 on membrane ruffling in RAW cells, we expressed inducible GTP binding–deficient versions of these proteins in RAW cells and selected stable clones. As a complementary approach, we also utilized the GAP domain of n-chimaerin (Chimaerin-GAP), previously shown to display GAP activity towards Rac1 and Cdc42 (22). Expression of Chimaerin-GAP would be expected to decrease the accumulation of GTP-bound (active) forms of Rac1 and Cdc42. We used a RAW cell clone (RAW LacR/FMLPR.2) that stably expressed both the human FMLP receptor and the Lac repressor protein as a recipient cell line. This line was used to generate further clones expressing Myc-tagged human Rac1 N17, Cdc42 N17, and Chimaerin-GAP. Between 50 and 200 clones expressing each construct were isolated. The clones varied in their “leakiness” (i.e., expression of the Myc epitope in the absence of the inducing agent, IPTG) and level of inducible expression. Therefore, we determined optimal conditions for the expression of the constructs, and selected three to six clones that displayed a high level of inducible expression for each construct. For example, in the absence of IPTG, minimal expression of the Myc epitope was detected, whereas IPTG caused a time-dependent appearance of fusion protein expression. The addition of zinc acetate and sodium butyrate further enhanced protein expression (Fig. 2 a). Fluorescence microscopy revealed a bimodal population of cells that either did or did not express the Myc epitope. In uninduced cells, <1% of the cells expressed the Myc epitope, whereas addition of IPTG, zinc, and butyrate induced expression of the Myc epitope in 20–60% of the total cell population. Clones were selected on the basis of demonstrating uniformly bright fluorescence in those individual cells that stained positive for the Myc epitope (i.e., cells within a selected clone showed a sharp contrast between Myc expression and a lack of expression, and the variation of intensities of Myc-positive cells was relatively small). We quantitated expression of the Myc-tagged fusion proteins on a single cell basis by measuring the fluorescence intensities of Myc-positive cells using microspectrofluorometry. Clones that inducibly expressed Rac1 N17 or Cdc42 N17 showed similar levels of expression, whereas Chimaerin-GAP consistently showed a greater level of expression on a single cell basis (P <0.05, when compared to any other clone; Fig. 2 b). In the 40–80% of the population that did not stain positive with mAb anti-Myc despite the presence of inducing agents, the mean fluorescence was similar to that of uninduced cells. Although we cannot explain why only a proportion of cells of a given clone expressed the fusion proteins, the nonexpressors of a given clone provided ideal controls to test the effects of fusion protein expression on cytoskeletal responses.

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