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Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis.

Srinivasan S, Wang F, Glavas S, Ott A, Hofmann F, Aktories K, Kalman D, Bourne HR - J. Cell Biol. (2003)

Bottom Line: Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3.Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac.We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology and the Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.

ABSTRACT
Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

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LT inhibits polarization, actin polymerization, chemotaxis, and PI(3,4,5)P3 production. (a) F-actin localization in differentiated HL-60 cells stimulated by a uniform concentration of fMLP (100 nM) without pretreatment (HL-60, left) or after pretreatment with LT (200 ng/ml for 24 h; +LT, right). Bar, 10 μm. (b) Fluorescence microscopic images of differentiated HL-60 cells stably expressing PH-Akt-GFP. Cells pretreated with LT (unstimulated, left) were exposed to a uniform concentration (100 nM) of fMLP for 2 min (+fMLP, middle) followed by stimulation with insulin (100 μg/ml for 2 min; +insulin, right). Bar, 10 μm. Note that 31 of 40 cells (80%) showed PH-Akt-GFP translocation in response to insulin; none of these cells showed translocation in response to fMLP. (c) Migration in the transwell assay of differentiated HL-60 cells treated (dotted line) or not treated (solid line) with LT. Cell counts indicate cells that crossed the transwell membrane.
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fig1: LT inhibits polarization, actin polymerization, chemotaxis, and PI(3,4,5)P3 production. (a) F-actin localization in differentiated HL-60 cells stimulated by a uniform concentration of fMLP (100 nM) without pretreatment (HL-60, left) or after pretreatment with LT (200 ng/ml for 24 h; +LT, right). Bar, 10 μm. (b) Fluorescence microscopic images of differentiated HL-60 cells stably expressing PH-Akt-GFP. Cells pretreated with LT (unstimulated, left) were exposed to a uniform concentration (100 nM) of fMLP for 2 min (+fMLP, middle) followed by stimulation with insulin (100 μg/ml for 2 min; +insulin, right). Bar, 10 μm. Note that 31 of 40 cells (80%) showed PH-Akt-GFP translocation in response to insulin; none of these cells showed translocation in response to fMLP. (c) Migration in the transwell assay of differentiated HL-60 cells treated (dotted line) or not treated (solid line) with LT. Cell counts indicate cells that crossed the transwell membrane.

Mentions: We reported previously (Servant et al., 2000) that Clostridium difficile toxin, which inhibits all known Rho GTPases (Sehr et al., 1998), blocks PI(3,4,5)P3 accumulation at the plasma membrane of differentiated HL-60 cells. Fig. 1 shows that Clostridium sordellii lethal toxin (LT) similarly prevented a neutrophil chemoattractant, formyl-methionine-leucine-phenylalanine (fMLP), from stimulating actin polymerization and formation of pseudopods (Fig. 1 a), and membrane translocation of a fluorescent PI(3,4,5)P3 probe (Fig. 1 b), the PH domain of Akt, tagged with GFP (PH-Akt-GFP). In addition, LT almost completely blocked fMLP-triggered migration across transwell filters (Fig. 1 c). LT ADP-ribosylates Rac and Cdc42 and inhibits their activities without affecting those of Rho (Just et al., 1996). In other experiments (unpublished data), a toxin that specifically inactivates Rho (C3 toxin) did not prevent PI(3,4,5)P3 accumulation. These effects do not reflect damage to the cells' intrinsic ability to accumulate PI(3,4,5)P3 as shown by the ability of the fMLP-unresponsive LT-treated cells to translocate PH-Akt-GFP to membranes in response to insulin (Fig. 1 b), a stimulus that activates PI(3,4,5)P3 accumulation by mechanisms that depend on tyrosine phosphorylation rather than on activation of a G protein–coupled receptor. These results confirm our previous inference (Servant et al., 2000) that Rho GTPases are required for polarity and PI(3,4,5)P3 accumulation and indicate that Rac or Cdc42, but not Rho, mediates both these responses to fMLP. We reported previously (Wang et al., 2002) that immunofluorescent staining with an mAb against PI(3,4,5)P3 showed a distribution identical to that of PH-Akt-GFP; this antibody shows 30-fold selectivity for PI(3,4,5)P3 versus PI(3,4)P2 (Chen et al., 2002).


Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis.

Srinivasan S, Wang F, Glavas S, Ott A, Hofmann F, Aktories K, Kalman D, Bourne HR - J. Cell Biol. (2003)

LT inhibits polarization, actin polymerization, chemotaxis, and PI(3,4,5)P3 production. (a) F-actin localization in differentiated HL-60 cells stimulated by a uniform concentration of fMLP (100 nM) without pretreatment (HL-60, left) or after pretreatment with LT (200 ng/ml for 24 h; +LT, right). Bar, 10 μm. (b) Fluorescence microscopic images of differentiated HL-60 cells stably expressing PH-Akt-GFP. Cells pretreated with LT (unstimulated, left) were exposed to a uniform concentration (100 nM) of fMLP for 2 min (+fMLP, middle) followed by stimulation with insulin (100 μg/ml for 2 min; +insulin, right). Bar, 10 μm. Note that 31 of 40 cells (80%) showed PH-Akt-GFP translocation in response to insulin; none of these cells showed translocation in response to fMLP. (c) Migration in the transwell assay of differentiated HL-60 cells treated (dotted line) or not treated (solid line) with LT. Cell counts indicate cells that crossed the transwell membrane.
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fig1: LT inhibits polarization, actin polymerization, chemotaxis, and PI(3,4,5)P3 production. (a) F-actin localization in differentiated HL-60 cells stimulated by a uniform concentration of fMLP (100 nM) without pretreatment (HL-60, left) or after pretreatment with LT (200 ng/ml for 24 h; +LT, right). Bar, 10 μm. (b) Fluorescence microscopic images of differentiated HL-60 cells stably expressing PH-Akt-GFP. Cells pretreated with LT (unstimulated, left) were exposed to a uniform concentration (100 nM) of fMLP for 2 min (+fMLP, middle) followed by stimulation with insulin (100 μg/ml for 2 min; +insulin, right). Bar, 10 μm. Note that 31 of 40 cells (80%) showed PH-Akt-GFP translocation in response to insulin; none of these cells showed translocation in response to fMLP. (c) Migration in the transwell assay of differentiated HL-60 cells treated (dotted line) or not treated (solid line) with LT. Cell counts indicate cells that crossed the transwell membrane.
Mentions: We reported previously (Servant et al., 2000) that Clostridium difficile toxin, which inhibits all known Rho GTPases (Sehr et al., 1998), blocks PI(3,4,5)P3 accumulation at the plasma membrane of differentiated HL-60 cells. Fig. 1 shows that Clostridium sordellii lethal toxin (LT) similarly prevented a neutrophil chemoattractant, formyl-methionine-leucine-phenylalanine (fMLP), from stimulating actin polymerization and formation of pseudopods (Fig. 1 a), and membrane translocation of a fluorescent PI(3,4,5)P3 probe (Fig. 1 b), the PH domain of Akt, tagged with GFP (PH-Akt-GFP). In addition, LT almost completely blocked fMLP-triggered migration across transwell filters (Fig. 1 c). LT ADP-ribosylates Rac and Cdc42 and inhibits their activities without affecting those of Rho (Just et al., 1996). In other experiments (unpublished data), a toxin that specifically inactivates Rho (C3 toxin) did not prevent PI(3,4,5)P3 accumulation. These effects do not reflect damage to the cells' intrinsic ability to accumulate PI(3,4,5)P3 as shown by the ability of the fMLP-unresponsive LT-treated cells to translocate PH-Akt-GFP to membranes in response to insulin (Fig. 1 b), a stimulus that activates PI(3,4,5)P3 accumulation by mechanisms that depend on tyrosine phosphorylation rather than on activation of a G protein–coupled receptor. These results confirm our previous inference (Servant et al., 2000) that Rho GTPases are required for polarity and PI(3,4,5)P3 accumulation and indicate that Rac or Cdc42, but not Rho, mediates both these responses to fMLP. We reported previously (Wang et al., 2002) that immunofluorescent staining with an mAb against PI(3,4,5)P3 showed a distribution identical to that of PH-Akt-GFP; this antibody shows 30-fold selectivity for PI(3,4,5)P3 versus PI(3,4)P2 (Chen et al., 2002).

Bottom Line: Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3.Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac.We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology and the Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.

ABSTRACT
Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

Show MeSH
Related in: MedlinePlus