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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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Inhibition of Cdc42 activity blocks migration toward a point source of chemoattractant. (a–c) Migration of cells toward a point source of fMLP (10 μM fMLP in a micropipette). Cells expressed PH-Akt-GFP alone (a) or in combination with Cdc42N17 (b) or WASpΔC (c). The top images show the morphology of a single cell by Nomarski microscopy at the indicated times after chemoattractant stimulation; the bottom images show spatial localization of PH-Akt-GFP in the same cell at the same time points. Arrows identify leading edges. Bars, 10 μm. The experiments in a and b are shown in videos 3 and 4, respectively, available at http://www.jcb.org/cgi/content/full/jcb.200208179/DC1. (d) Outlines of migrating cells observed after exposure to an fMLP-containing micropipette (locations indicated by asterisks). As in Fig. 5 c, each set of outlines represents a cell observed at 1-min intervals (denoted by colors as indicated), from 0 to 3 min after exposure to the micropipette. Small circles in each outline represent the center of a PH-Akt-GFP–containing lamella at the cell periphery.
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fig6: Inhibition of Cdc42 activity blocks migration toward a point source of chemoattractant. (a–c) Migration of cells toward a point source of fMLP (10 μM fMLP in a micropipette). Cells expressed PH-Akt-GFP alone (a) or in combination with Cdc42N17 (b) or WASpΔC (c). The top images show the morphology of a single cell by Nomarski microscopy at the indicated times after chemoattractant stimulation; the bottom images show spatial localization of PH-Akt-GFP in the same cell at the same time points. Arrows identify leading edges. Bars, 10 μm. The experiments in a and b are shown in videos 3 and 4, respectively, available at http://www.jcb.org/cgi/content/full/jcb.200208179/DC1. (d) Outlines of migrating cells observed after exposure to an fMLP-containing micropipette (locations indicated by asterisks). As in Fig. 5 c, each set of outlines represents a cell observed at 1-min intervals (denoted by colors as indicated), from 0 to 3 min after exposure to the micropipette. Small circles in each outline represent the center of a PH-Akt-GFP–containing lamella at the cell periphery.

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)

Inhibition of Cdc42 activity blocks migration toward a point source of chemoattractant. (a–c) Migration of cells toward a point source of fMLP (10 μM fMLP in a micropipette). Cells expressed PH-Akt-GFP alone (a) or in combination with Cdc42N17 (b) or WASpΔC (c). The top images show the morphology of a single cell by Nomarski microscopy at the indicated times after chemoattractant stimulation; the bottom images show spatial localization of PH-Akt-GFP in the same cell at the same time points. Arrows identify leading edges. Bars, 10 μm. The experiments in a and b are shown in videos 3 and 4, respectively, available at http://www.jcb.org/cgi/content/full/jcb.200208179/DC1. (d) Outlines of migrating cells observed after exposure to an fMLP-containing micropipette (locations indicated by asterisks). As in Fig. 5 c, each set of outlines represents a cell observed at 1-min intervals (denoted by colors as indicated), from 0 to 3 min after exposure to the micropipette. Small circles in each outline represent the center of a PH-Akt-GFP–containing lamella at the cell periphery.
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Related In: Results  -  Collection

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fig6: Inhibition of Cdc42 activity blocks migration toward a point source of chemoattractant. (a–c) Migration of cells toward a point source of fMLP (10 μM fMLP in a micropipette). Cells expressed PH-Akt-GFP alone (a) or in combination with Cdc42N17 (b) or WASpΔC (c). The top images show the morphology of a single cell by Nomarski microscopy at the indicated times after chemoattractant stimulation; the bottom images show spatial localization of PH-Akt-GFP in the same cell at the same time points. Arrows identify leading edges. Bars, 10 μm. The experiments in a and b are shown in videos 3 and 4, respectively, available at http://www.jcb.org/cgi/content/full/jcb.200208179/DC1. (d) Outlines of migrating cells observed after exposure to an fMLP-containing micropipette (locations indicated by asterisks). As in Fig. 5 c, each set of outlines represents a cell observed at 1-min intervals (denoted by colors as indicated), from 0 to 3 min after exposure to the micropipette. Small circles in each outline represent the center of a PH-Akt-GFP–containing lamella at the cell periphery.
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