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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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Actin polymerization is necessary for Rac-dependent PI(3,4,5)P3 accumulation. (a) Spatial distribution of PH-Akt-GFP (top) or PAK-PBD-YFP (bottom) in cells expressing RacV12 before and after exposure to latrunculin B (20 μg/ml) for the indicated times. (b) PAK-PBD-YFP and myc-tagged RacV12 show persistent colocalization at the membrane even after treatment with latrunculin B for 10 min. Bars, 10 μm.
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fig4: Actin polymerization is necessary for Rac-dependent PI(3,4,5)P3 accumulation. (a) Spatial distribution of PH-Akt-GFP (top) or PAK-PBD-YFP (bottom) in cells expressing RacV12 before and after exposure to latrunculin B (20 μg/ml) for the indicated times. (b) PAK-PBD-YFP and myc-tagged RacV12 show persistent colocalization at the membrane even after treatment with latrunculin B for 10 min. Bars, 10 μm.

Mentions: To ask whether Rac-stimulated PI(3,4,5)P3 accumulation does depend on actin polymerization, we tested the effect of exposing RacV12-expressing cells to latrunculin in the absence of fMLP stimulation. Latrunculin caused PH-Akt-GFP to translocate back from plasma membrane to cytosol (Fig. 4 a). This effect was reproducible: 72% of RacV12-expressing control cells examined showed PH-Akt-GFP located at the plasma membrane, whereas after treatment with latrunculin only 16% of RacV12-expressing cells did so (Table I). In contrast, latrunculin did not cause PAK-PBD-YFP, the probe for activated Rac, to return to the cytoplasm (Fig. 4 a). Instead, PAK-PBD-YFP remained at the membrane where it colocalized with myc-tagged RacV12 (Fig. 4 b). We infer that, at least in the absence of fMLP, stimulation of membrane PI(3,4,5)P3 accumulation by Rac is mediated by (or depends on) actin polymerization.


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)

Actin polymerization is necessary for Rac-dependent PI(3,4,5)P3 accumulation. (a) Spatial distribution of PH-Akt-GFP (top) or PAK-PBD-YFP (bottom) in cells expressing RacV12 before and after exposure to latrunculin B (20 μg/ml) for the indicated times. (b) PAK-PBD-YFP and myc-tagged RacV12 show persistent colocalization at the membrane even after treatment with latrunculin B for 10 min. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172671&req=5

fig4: Actin polymerization is necessary for Rac-dependent PI(3,4,5)P3 accumulation. (a) Spatial distribution of PH-Akt-GFP (top) or PAK-PBD-YFP (bottom) in cells expressing RacV12 before and after exposure to latrunculin B (20 μg/ml) for the indicated times. (b) PAK-PBD-YFP and myc-tagged RacV12 show persistent colocalization at the membrane even after treatment with latrunculin B for 10 min. Bars, 10 μm.
Mentions: To ask whether Rac-stimulated PI(3,4,5)P3 accumulation does depend on actin polymerization, we tested the effect of exposing RacV12-expressing cells to latrunculin in the absence of fMLP stimulation. Latrunculin caused PH-Akt-GFP to translocate back from plasma membrane to cytosol (Fig. 4 a). This effect was reproducible: 72% of RacV12-expressing control cells examined showed PH-Akt-GFP located at the plasma membrane, whereas after treatment with latrunculin only 16% of RacV12-expressing cells did so (Table I). In contrast, latrunculin did not cause PAK-PBD-YFP, the probe for activated Rac, to return to the cytoplasm (Fig. 4 a). Instead, PAK-PBD-YFP remained at the membrane where it colocalized with myc-tagged RacV12 (Fig. 4 b). We infer that, at least in the absence of fMLP, stimulation of membrane PI(3,4,5)P3 accumulation by Rac is mediated by (or depends on) actin polymerization.

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