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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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Model for the spatial and temporal regulation of Ras-induced chemotaxis. A model illustrating the intracellular signaling leading to local PI(3,4,5)P3 production is shown. (A) Resting cells with a basal level of PI3K at the plasma membrane. (B) The chemoattractant locally activates Ras at the presumptive leading edge (site closest to the chemoattractant source), where Ras then locally activates PI3K. There is a local polymerization of F-actin at the presumptive leading edge, which is partially Ras/PI3K independent, presumably controlled by Rho GTPase, WASP/SCAR, and Arp2/3. Our data suggest that the F-actin mediates PI3K translocation. (C) Locally produced PI(3,4,5)P3 induces further F-actin polymerization by activating downstream effectors, which would enhance the recruitment of PI3K to the membrane.
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fig8: Model for the spatial and temporal regulation of Ras-induced chemotaxis. A model illustrating the intracellular signaling leading to local PI(3,4,5)P3 production is shown. (A) Resting cells with a basal level of PI3K at the plasma membrane. (B) The chemoattractant locally activates Ras at the presumptive leading edge (site closest to the chemoattractant source), where Ras then locally activates PI3K. There is a local polymerization of F-actin at the presumptive leading edge, which is partially Ras/PI3K independent, presumably controlled by Rho GTPase, WASP/SCAR, and Arp2/3. Our data suggest that the F-actin mediates PI3K translocation. (C) Locally produced PI(3,4,5)P3 induces further F-actin polymerization by activating downstream effectors, which would enhance the recruitment of PI3K to the membrane.

Mentions: When a chemoattractant gradient is applied to cells, both PI(3,4,5)P3 accumulation and Ras activation occur locally at the site on the plasma membrane closest to the micropipette. We suggest that the localized increase of PI(3,4,5)P3 is the result of coordination between F-actin–independent local Ras activation and F-actin–driven PI3K localization (Fig. 8). The kinetics of Ras activation, F-actin polymerization, and PI3K translocation are similar, although our detailed analysis suggests that Ras activation (membrane localization of the GFP-RBD reporter) slightly precedes the localization of N-PI3K1. Strikingly, we found that translocation of N-PI3K1 (and full-length PI3K1 and PI3K2) is regulated by F-actin polymerization. We have shown that unstimulated cells have a low, basal level of PI3K on the plasma membrane.


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)

Model for the spatial and temporal regulation of Ras-induced chemotaxis. A model illustrating the intracellular signaling leading to local PI(3,4,5)P3 production is shown. (A) Resting cells with a basal level of PI3K at the plasma membrane. (B) The chemoattractant locally activates Ras at the presumptive leading edge (site closest to the chemoattractant source), where Ras then locally activates PI3K. There is a local polymerization of F-actin at the presumptive leading edge, which is partially Ras/PI3K independent, presumably controlled by Rho GTPase, WASP/SCAR, and Arp2/3. Our data suggest that the F-actin mediates PI3K translocation. (C) Locally produced PI(3,4,5)P3 induces further F-actin polymerization by activating downstream effectors, which would enhance the recruitment of PI3K to the membrane.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172490&req=5

fig8: Model for the spatial and temporal regulation of Ras-induced chemotaxis. A model illustrating the intracellular signaling leading to local PI(3,4,5)P3 production is shown. (A) Resting cells with a basal level of PI3K at the plasma membrane. (B) The chemoattractant locally activates Ras at the presumptive leading edge (site closest to the chemoattractant source), where Ras then locally activates PI3K. There is a local polymerization of F-actin at the presumptive leading edge, which is partially Ras/PI3K independent, presumably controlled by Rho GTPase, WASP/SCAR, and Arp2/3. Our data suggest that the F-actin mediates PI3K translocation. (C) Locally produced PI(3,4,5)P3 induces further F-actin polymerization by activating downstream effectors, which would enhance the recruitment of PI3K to the membrane.
Mentions: When a chemoattractant gradient is applied to cells, both PI(3,4,5)P3 accumulation and Ras activation occur locally at the site on the plasma membrane closest to the micropipette. We suggest that the localized increase of PI(3,4,5)P3 is the result of coordination between F-actin–independent local Ras activation and F-actin–driven PI3K localization (Fig. 8). The kinetics of Ras activation, F-actin polymerization, and PI3K translocation are similar, although our detailed analysis suggests that Ras activation (membrane localization of the GFP-RBD reporter) slightly precedes the localization of N-PI3K1. Strikingly, we found that translocation of N-PI3K1 (and full-length PI3K1 and PI3K2) is regulated by F-actin polymerization. We have shown that unstimulated cells have a low, basal level of PI3K on the plasma membrane.

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
Related in: MedlinePlus