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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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Spatial-temporal activation of Ras during chemotaxis. (A and B) The localization of GFP-RBD in wild-type vegetative cells (A) or aggregation-competent cells (B) was imaged. Translocation of GFP-RBD was imaged after stimulation with cAMP as described previously (Funamoto et al., 2001). (C) Translocation kinetics of GFP-tagged PhdA, CRAC-PH, N-PI3K1, and RBD in wild-type cells were obtained from time-lapse recordings. The graphs represent an average of data of movies taken from at least three separate experiments. The fluorescence intensity of membrane-localized GFP fusion protein was quantitated as Et as defined in Materials and methods. (D) Translocation of indicated GFP proteins in pi3k1/2  cells or wild-type cells treated with 50 μM LY294002 for 20 min before cAMP stimulation. (E–I) Fluorescent images of GFP-RBD expressing wild-type cells (E and F), pi3k1/2  cells (G), or pten  and myr-PI3K expressing cells (I) chemotaxing in a gradient of chemoattractant. An asterisk indicates the position of the micropipette. Fluorescent images of GFP-RBD and myr-PI3K1–expressing pi3k1/2  cells (H, left) or GFP-RBD-expressing pten  cells (H, right) are shown. The sites producing multiple pseudopodia are marked with arrowheads.
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fig3: Spatial-temporal activation of Ras during chemotaxis. (A and B) The localization of GFP-RBD in wild-type vegetative cells (A) or aggregation-competent cells (B) was imaged. Translocation of GFP-RBD was imaged after stimulation with cAMP as described previously (Funamoto et al., 2001). (C) Translocation kinetics of GFP-tagged PhdA, CRAC-PH, N-PI3K1, and RBD in wild-type cells were obtained from time-lapse recordings. The graphs represent an average of data of movies taken from at least three separate experiments. The fluorescence intensity of membrane-localized GFP fusion protein was quantitated as Et as defined in Materials and methods. (D) Translocation of indicated GFP proteins in pi3k1/2 cells or wild-type cells treated with 50 μM LY294002 for 20 min before cAMP stimulation. (E–I) Fluorescent images of GFP-RBD expressing wild-type cells (E and F), pi3k1/2 cells (G), or pten and myr-PI3K expressing cells (I) chemotaxing in a gradient of chemoattractant. An asterisk indicates the position of the micropipette. Fluorescent images of GFP-RBD and myr-PI3K1–expressing pi3k1/2 cells (H, left) or GFP-RBD-expressing pten cells (H, right) are shown. The sites producing multiple pseudopodia are marked with arrowheads.

Mentions: To determine the spatial-temporal kinetics of Ras activation in living cells, we used the GFP-tagged RBD of Raf1, which binds to and thus should monitor Ras-GTP levels and its localization in cells (Chiu et al., 2002; Hancock, 2003). We investigated the GFP-RBD localization in randomly moving vegetative cells. As illustrated in Fig. 3 A, resting vegetative cells display an even cytoplasmic fluorescent signal with labeling of regions of membrane ruffling and circular, “crown-like” structures, which may be the precursors of macropinosomes. These are the regions in which F-actin and PI(3,4,5)P3 accumulate and to which PI3K specifically translocates (Parent et al., 1998; Funamoto et al., 2001, 2002). In aggregation-competent cells, which are differentiated by cAMP pulsing and competent to chemotax to cAMP, PH domain-containing proteins (e.g., CRAC, Akt/PKB, and PhdA) and PI3K rapidly translocate to the plasma membrane in response to global stimulation with a chemoattractant (Parent et al., 1998; Meili et al., 1999; Funamoto et al., 2002). We measured the kinetics of PH domain and PI3K cortical localizations using a faster and more sensitive camera than used previously, allowing us to see differences in the initial steps of the two responses (Funamoto et al., 2002). As shown in Fig. 3 C, the translocation of PI3K occurs faster than that of the PH domain, whereas the loss of PI3K from the plasma membrane occurs more slowly than that of the PH domain. For this work, we use the NH2-terminal domain of PI3K1 (N-PI3K1) that does not contain the RBD and is necessary and sufficient for PI3K localization. This reporter exhibits the same subcellular localization and kinetics of localization as GFP-PI3K (Funamoto et al., 2002). Interestingly, GFP-RBD also rapidly and transiently localizes to the plasma membrane in response to global stimulation (Fig. 3 B and Video 1, available at http:www.jcb.org/cgi/content/full/jcb.200406177/DC1). The signal intensity of GFP-RBD was significantly weaker than that of the PH domain-containing proteins, possibly because the potential binding sites for PH domains (PI(3,4,5)P3/PI(3,4)P2) are amplified relative to Ras-GTP by the catalytic activity of PI3K, thus providing an amplified signal, and/or because of our inability to obtain strains expressing high levels of RBD-GFP compared with the levels of expression of GFP fusions of PH domains and N-PI3K. The weaker signal could also be caused by the affinity of the Raf1 RBD for Ras-GTP in an in vivo cellular context.


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)

Spatial-temporal activation of Ras during chemotaxis. (A and B) The localization of GFP-RBD in wild-type vegetative cells (A) or aggregation-competent cells (B) was imaged. Translocation of GFP-RBD was imaged after stimulation with cAMP as described previously (Funamoto et al., 2001). (C) Translocation kinetics of GFP-tagged PhdA, CRAC-PH, N-PI3K1, and RBD in wild-type cells were obtained from time-lapse recordings. The graphs represent an average of data of movies taken from at least three separate experiments. The fluorescence intensity of membrane-localized GFP fusion protein was quantitated as Et as defined in Materials and methods. (D) Translocation of indicated GFP proteins in pi3k1/2  cells or wild-type cells treated with 50 μM LY294002 for 20 min before cAMP stimulation. (E–I) Fluorescent images of GFP-RBD expressing wild-type cells (E and F), pi3k1/2  cells (G), or pten  and myr-PI3K expressing cells (I) chemotaxing in a gradient of chemoattractant. An asterisk indicates the position of the micropipette. Fluorescent images of GFP-RBD and myr-PI3K1–expressing pi3k1/2  cells (H, left) or GFP-RBD-expressing pten  cells (H, right) are shown. The sites producing multiple pseudopodia are marked with arrowheads.
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Related In: Results  -  Collection

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fig3: Spatial-temporal activation of Ras during chemotaxis. (A and B) The localization of GFP-RBD in wild-type vegetative cells (A) or aggregation-competent cells (B) was imaged. Translocation of GFP-RBD was imaged after stimulation with cAMP as described previously (Funamoto et al., 2001). (C) Translocation kinetics of GFP-tagged PhdA, CRAC-PH, N-PI3K1, and RBD in wild-type cells were obtained from time-lapse recordings. The graphs represent an average of data of movies taken from at least three separate experiments. The fluorescence intensity of membrane-localized GFP fusion protein was quantitated as Et as defined in Materials and methods. (D) Translocation of indicated GFP proteins in pi3k1/2 cells or wild-type cells treated with 50 μM LY294002 for 20 min before cAMP stimulation. (E–I) Fluorescent images of GFP-RBD expressing wild-type cells (E and F), pi3k1/2 cells (G), or pten and myr-PI3K expressing cells (I) chemotaxing in a gradient of chemoattractant. An asterisk indicates the position of the micropipette. Fluorescent images of GFP-RBD and myr-PI3K1–expressing pi3k1/2 cells (H, left) or GFP-RBD-expressing pten cells (H, right) are shown. The sites producing multiple pseudopodia are marked with arrowheads.
Mentions: To determine the spatial-temporal kinetics of Ras activation in living cells, we used the GFP-tagged RBD of Raf1, which binds to and thus should monitor Ras-GTP levels and its localization in cells (Chiu et al., 2002; Hancock, 2003). We investigated the GFP-RBD localization in randomly moving vegetative cells. As illustrated in Fig. 3 A, resting vegetative cells display an even cytoplasmic fluorescent signal with labeling of regions of membrane ruffling and circular, “crown-like” structures, which may be the precursors of macropinosomes. These are the regions in which F-actin and PI(3,4,5)P3 accumulate and to which PI3K specifically translocates (Parent et al., 1998; Funamoto et al., 2001, 2002). In aggregation-competent cells, which are differentiated by cAMP pulsing and competent to chemotax to cAMP, PH domain-containing proteins (e.g., CRAC, Akt/PKB, and PhdA) and PI3K rapidly translocate to the plasma membrane in response to global stimulation with a chemoattractant (Parent et al., 1998; Meili et al., 1999; Funamoto et al., 2002). We measured the kinetics of PH domain and PI3K cortical localizations using a faster and more sensitive camera than used previously, allowing us to see differences in the initial steps of the two responses (Funamoto et al., 2002). As shown in Fig. 3 C, the translocation of PI3K occurs faster than that of the PH domain, whereas the loss of PI3K from the plasma membrane occurs more slowly than that of the PH domain. For this work, we use the NH2-terminal domain of PI3K1 (N-PI3K1) that does not contain the RBD and is necessary and sufficient for PI3K localization. This reporter exhibits the same subcellular localization and kinetics of localization as GFP-PI3K (Funamoto et al., 2002). Interestingly, GFP-RBD also rapidly and transiently localizes to the plasma membrane in response to global stimulation (Fig. 3 B and Video 1, available at http:www.jcb.org/cgi/content/full/jcb.200406177/DC1). The signal intensity of GFP-RBD was significantly weaker than that of the PH domain-containing proteins, possibly because the potential binding sites for PH domains (PI(3,4,5)P3/PI(3,4)P2) are amplified relative to Ras-GTP by the catalytic activity of PI3K, thus providing an amplified signal, and/or because of our inability to obtain strains expressing high levels of RBD-GFP compared with the levels of expression of GFP fusions of PH domains and N-PI3K. The weaker signal could also be caused by the affinity of the Raf1 RBD for Ras-GTP in an in vivo cellular context.

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.

Show MeSH