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Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.

Sasaki AT, Chun C, Takeda K, Firtel RA - J. Cell Biol. (2004)

Bottom Line: Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass.These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis.A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P(3), the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P(3) asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

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Differential regulation between PI3K translocation and Ras activation. (A and B) Effect of LatA on the translocation of GFP-tagged PhdA-, N-PI3K1–, and RBD-expressing wild-type cells and pten  cells. Asterisk indicates the position of the micropipette (B). These images were captured ∼30 s after changing the micropipette position. The behaviors were consistently observed over five independent sessions. Translocation kinetics of RBD were obtained from a time-lapse recording of GFP-RBD–expressing wild-type cells. Fluorescence intensities of the upper and lower plasma membranes were quantitated as Et (see Materials and methods). (C) Effect of LatA on Akt/PKB activation. Akt/PKB assays were performed as described in Fig. 1. Data are representative of at least three independent experiments. (D) N-PI3K1 is recruited into the Triton X-100–resistant cytoskeleton in response to chemoattractant stimulation. Cytoskeletal fractions (see Materials and methods) were subjected to SDS-PAGE. Actin and N-PI3K1 recruitment were assessed with Coomassie stain or anti-GFP antibody, respectively. Ras activation was assayed using an aliquoted lysate. Graph shows an average of the results that were obtained from four independent experiments. (E) Fluorescent images of GFP-tagged N-PI3K1–expressing wild-type cells. Cells were treated with (right) or without (left) 0.01% Triton X-100 before fixation by 3.7% formaldehyde.
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fig4: Differential regulation between PI3K translocation and Ras activation. (A and B) Effect of LatA on the translocation of GFP-tagged PhdA-, N-PI3K1–, and RBD-expressing wild-type cells and pten cells. Asterisk indicates the position of the micropipette (B). These images were captured ∼30 s after changing the micropipette position. The behaviors were consistently observed over five independent sessions. Translocation kinetics of RBD were obtained from a time-lapse recording of GFP-RBD–expressing wild-type cells. Fluorescence intensities of the upper and lower plasma membranes were quantitated as Et (see Materials and methods). (C) Effect of LatA on Akt/PKB activation. Akt/PKB assays were performed as described in Fig. 1. Data are representative of at least three independent experiments. (D) N-PI3K1 is recruited into the Triton X-100–resistant cytoskeleton in response to chemoattractant stimulation. Cytoskeletal fractions (see Materials and methods) were subjected to SDS-PAGE. Actin and N-PI3K1 recruitment were assessed with Coomassie stain or anti-GFP antibody, respectively. Ras activation was assayed using an aliquoted lysate. Graph shows an average of the results that were obtained from four independent experiments. (E) Fluorescent images of GFP-tagged N-PI3K1–expressing wild-type cells. Cells were treated with (right) or without (left) 0.01% Triton X-100 before fixation by 3.7% formaldehyde.

Mentions: In response to chemoattractant stimulation, Dictyostelium and mammalian cell types exhibit a rapid burst of F-actin polymerization, which is followed by a rapid decrease to a near-basal level and a subsequent slower rise to a peak level ∼1/3 as high as the initial peak (Hall et al., 1988). The initial F-actin peak is similar in timing to Ras activation and PI3K translocation. Therefore, we examined if F-actin polymerization was needed to locally activate leading-edge signaling events using the F-actin polymerization inhibitor latrunculin A (LatA). After LatA treatment, cells became progressively rounder and stopped moving (Parent et al., 1998). Consistent with previous findings, under these conditions, there was a reduced amount of F-actin associated with the Triton X-100–insoluble cortical fraction and chemoattractant stimulation did not induce F-actin polymerization (Fig. 4 D). As in previous work (Parent et al., 1998), a global stimulation of LatA-treated cells resulted in a rapid cortical localization of the PH-domain-containing protein (Fig. 4 A).


Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.

Sasaki AT, Chun C, Takeda K, Firtel RA - J. Cell Biol. (2004)

Differential regulation between PI3K translocation and Ras activation. (A and B) Effect of LatA on the translocation of GFP-tagged PhdA-, N-PI3K1–, and RBD-expressing wild-type cells and pten  cells. Asterisk indicates the position of the micropipette (B). These images were captured ∼30 s after changing the micropipette position. The behaviors were consistently observed over five independent sessions. Translocation kinetics of RBD were obtained from a time-lapse recording of GFP-RBD–expressing wild-type cells. Fluorescence intensities of the upper and lower plasma membranes were quantitated as Et (see Materials and methods). (C) Effect of LatA on Akt/PKB activation. Akt/PKB assays were performed as described in Fig. 1. Data are representative of at least three independent experiments. (D) N-PI3K1 is recruited into the Triton X-100–resistant cytoskeleton in response to chemoattractant stimulation. Cytoskeletal fractions (see Materials and methods) were subjected to SDS-PAGE. Actin and N-PI3K1 recruitment were assessed with Coomassie stain or anti-GFP antibody, respectively. Ras activation was assayed using an aliquoted lysate. Graph shows an average of the results that were obtained from four independent experiments. (E) Fluorescent images of GFP-tagged N-PI3K1–expressing wild-type cells. Cells were treated with (right) or without (left) 0.01% Triton X-100 before fixation by 3.7% formaldehyde.
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fig4: Differential regulation between PI3K translocation and Ras activation. (A and B) Effect of LatA on the translocation of GFP-tagged PhdA-, N-PI3K1–, and RBD-expressing wild-type cells and pten cells. Asterisk indicates the position of the micropipette (B). These images were captured ∼30 s after changing the micropipette position. The behaviors were consistently observed over five independent sessions. Translocation kinetics of RBD were obtained from a time-lapse recording of GFP-RBD–expressing wild-type cells. Fluorescence intensities of the upper and lower plasma membranes were quantitated as Et (see Materials and methods). (C) Effect of LatA on Akt/PKB activation. Akt/PKB assays were performed as described in Fig. 1. Data are representative of at least three independent experiments. (D) N-PI3K1 is recruited into the Triton X-100–resistant cytoskeleton in response to chemoattractant stimulation. Cytoskeletal fractions (see Materials and methods) were subjected to SDS-PAGE. Actin and N-PI3K1 recruitment were assessed with Coomassie stain or anti-GFP antibody, respectively. Ras activation was assayed using an aliquoted lysate. Graph shows an average of the results that were obtained from four independent experiments. (E) Fluorescent images of GFP-tagged N-PI3K1–expressing wild-type cells. Cells were treated with (right) or without (left) 0.01% Triton X-100 before fixation by 3.7% formaldehyde.
Mentions: In response to chemoattractant stimulation, Dictyostelium and mammalian cell types exhibit a rapid burst of F-actin polymerization, which is followed by a rapid decrease to a near-basal level and a subsequent slower rise to a peak level ∼1/3 as high as the initial peak (Hall et al., 1988). The initial F-actin peak is similar in timing to Ras activation and PI3K translocation. Therefore, we examined if F-actin polymerization was needed to locally activate leading-edge signaling events using the F-actin polymerization inhibitor latrunculin A (LatA). After LatA treatment, cells became progressively rounder and stopped moving (Parent et al., 1998). Consistent with previous findings, under these conditions, there was a reduced amount of F-actin associated with the Triton X-100–insoluble cortical fraction and chemoattractant stimulation did not induce F-actin polymerization (Fig. 4 D). As in previous work (Parent et al., 1998), a global stimulation of LatA-treated cells resulted in a rapid cortical localization of the PH-domain-containing protein (Fig. 4 A).

Bottom Line: Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass.These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis.A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P(3), the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P(3) asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

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