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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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PAK-PBD-YFP reflects localization of activated Rac at the plasma membrane but not that of activated Cdc42. Localization of PAK-PBD-YFP in cells exposed for 3 min to 100 nM fMLP (a–c); in addition to PAK-PBD-YFP, the cell in b coexpressed RacN17 and the cell in c coexpressed Cdc42N17. Cells in d–f and g–i expressed constitutively active Rac or Cdc42, respectively; after 3 min exposure to fMLP, these cells were fixed and imaged for PAK-PBD-YFP fluorescence or immunofluorescence of the myc-tagged Rho GTPase as indicated. Bar, 10 μm.
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fig3: PAK-PBD-YFP reflects localization of activated Rac at the plasma membrane but not that of activated Cdc42. Localization of PAK-PBD-YFP in cells exposed for 3 min to 100 nM fMLP (a–c); in addition to PAK-PBD-YFP, the cell in b coexpressed RacN17 and the cell in c coexpressed Cdc42N17. Cells in d–f and g–i expressed constitutively active Rac or Cdc42, respectively; after 3 min exposure to fMLP, these cells were fixed and imaged for PAK-PBD-YFP fluorescence or immunofluorescence of the myc-tagged Rho GTPase as indicated. Bar, 10 μm.

Mentions: We developed a transient transfection protocol that allowed us to identify distinct roles of Rac and Cdc42 in regulating polarity and PI(3,4,5)P3 accumulation, in determining subcellular distribution of activated Rac, and in directing cell migration (Figs. 2–6). Dominant interfering mutant proteins and fluorescent probes were transiently expressed in differentiated HL-60 cells, with transfection efficiencies that varied from 10 to 20%. In populations cotransfected with two constructs, >80% of cells expressing one also expressed the other (for details see Materials and methods). Although they migrate slightly less rapidly (see Materials and methods) than do cells stably expressing PH-Akt-GFP (Wang et al., 2002), cells transiently expressing the lipid probe showed unchanged responses to fMLP, including morphologic polarity, formation of actin polymers, directed motility, and asymmetric accumulation of the probe for activated Rac (Figs. 2, 3 a, 5 a, and 6 a).


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)

PAK-PBD-YFP reflects localization of activated Rac at the plasma membrane but not that of activated Cdc42. Localization of PAK-PBD-YFP in cells exposed for 3 min to 100 nM fMLP (a–c); in addition to PAK-PBD-YFP, the cell in b coexpressed RacN17 and the cell in c coexpressed Cdc42N17. Cells in d–f and g–i expressed constitutively active Rac or Cdc42, respectively; after 3 min exposure to fMLP, these cells were fixed and imaged for PAK-PBD-YFP fluorescence or immunofluorescence of the myc-tagged Rho GTPase as indicated. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: PAK-PBD-YFP reflects localization of activated Rac at the plasma membrane but not that of activated Cdc42. Localization of PAK-PBD-YFP in cells exposed for 3 min to 100 nM fMLP (a–c); in addition to PAK-PBD-YFP, the cell in b coexpressed RacN17 and the cell in c coexpressed Cdc42N17. Cells in d–f and g–i expressed constitutively active Rac or Cdc42, respectively; after 3 min exposure to fMLP, these cells were fixed and imaged for PAK-PBD-YFP fluorescence or immunofluorescence of the myc-tagged Rho GTPase as indicated. Bar, 10 μm.
Mentions: We developed a transient transfection protocol that allowed us to identify distinct roles of Rac and Cdc42 in regulating polarity and PI(3,4,5)P3 accumulation, in determining subcellular distribution of activated Rac, and in directing cell migration (Figs. 2–6). Dominant interfering mutant proteins and fluorescent probes were transiently expressed in differentiated HL-60 cells, with transfection efficiencies that varied from 10 to 20%. In populations cotransfected with two constructs, >80% of cells expressing one also expressed the other (for details see Materials and methods). Although they migrate slightly less rapidly (see Materials and methods) than do cells stably expressing PH-Akt-GFP (Wang et al., 2002), cells transiently expressing the lipid probe showed unchanged responses to fMLP, including morphologic polarity, formation of actin polymers, directed motility, and asymmetric accumulation of the probe for activated Rac (Figs. 2, 3 a, 5 a, and 6 a).

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