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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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Ras signaling regulates proper chemotaxis. (A–F) Time-lapse recording of chemotaxis of indicated strains pulsed with cAMP for 6 h. An asterisk indicates the position of a micropipette containing 150 μM cAMP. Tracing of the chemotaxis of individual cells is shown. The expression level of myc-tagged RasGS17N in aleA  cells was monitored by immunofluorescence (E, inset). (G) Analysis of chemotaxis using DIAS software (Soll and Voss, 1998). Speed refers to the speed of the cell's centroid movement along the total path. Directionality is a measure of how straight the cells move. Direction change (Dire Ch) is a measure of the number and frequency of turns the cell makes. Roundness is an indication of the polarity of the cells.
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fig6: Ras signaling regulates proper chemotaxis. (A–F) Time-lapse recording of chemotaxis of indicated strains pulsed with cAMP for 6 h. An asterisk indicates the position of a micropipette containing 150 μM cAMP. Tracing of the chemotaxis of individual cells is shown. The expression level of myc-tagged RasGS17N in aleA cells was monitored by immunofluorescence (E, inset). (G) Analysis of chemotaxis using DIAS software (Soll and Voss, 1998). Speed refers to the speed of the cell's centroid movement along the total path. Directionality is a measure of how straight the cells move. Direction change (Dire Ch) is a measure of the number and frequency of turns the cell makes. Roundness is an indication of the polarity of the cells.

Mentions: Our previous findings that a functional RBD is required for Dictyostelium PI3K activation (Funamoto et al., 2002) and our observations here that Ras is activated at the leading edge suggest that RasG and Ras proteins of related function may be required for directional sensing. Further, several studies have implicated Ras in chemotaxis: rasG cells have a slight loss of motility (Tuxworth et al., 1997; Lim et al., 2002), and a mutation of the RasG-GTP binding protein RIP3 exhibits chemotaxis defects (Lee et al., 1999). Fig. 6 (A, B, and G; and Videos 5 and 6, available at http:www.jcb.org/cgi/content/full/jcb.200406177/DC1) extends these studies and shows that rasG cells, compared with wild-type cells, exhibit reduced cell polarity and directionality (persistent movement toward the chemoattractant source).


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)

Ras signaling regulates proper chemotaxis. (A–F) Time-lapse recording of chemotaxis of indicated strains pulsed with cAMP for 6 h. An asterisk indicates the position of a micropipette containing 150 μM cAMP. Tracing of the chemotaxis of individual cells is shown. The expression level of myc-tagged RasGS17N in aleA  cells was monitored by immunofluorescence (E, inset). (G) Analysis of chemotaxis using DIAS software (Soll and Voss, 1998). Speed refers to the speed of the cell's centroid movement along the total path. Directionality is a measure of how straight the cells move. Direction change (Dire Ch) is a measure of the number and frequency of turns the cell makes. Roundness is an indication of the polarity of the cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172490&req=5

fig6: Ras signaling regulates proper chemotaxis. (A–F) Time-lapse recording of chemotaxis of indicated strains pulsed with cAMP for 6 h. An asterisk indicates the position of a micropipette containing 150 μM cAMP. Tracing of the chemotaxis of individual cells is shown. The expression level of myc-tagged RasGS17N in aleA cells was monitored by immunofluorescence (E, inset). (G) Analysis of chemotaxis using DIAS software (Soll and Voss, 1998). Speed refers to the speed of the cell's centroid movement along the total path. Directionality is a measure of how straight the cells move. Direction change (Dire Ch) is a measure of the number and frequency of turns the cell makes. Roundness is an indication of the polarity of the cells.
Mentions: Our previous findings that a functional RBD is required for Dictyostelium PI3K activation (Funamoto et al., 2002) and our observations here that Ras is activated at the leading edge suggest that RasG and Ras proteins of related function may be required for directional sensing. Further, several studies have implicated Ras in chemotaxis: rasG cells have a slight loss of motility (Tuxworth et al., 1997; Lim et al., 2002), and a mutation of the RasG-GTP binding protein RIP3 exhibits chemotaxis defects (Lee et al., 1999). Fig. 6 (A, B, and G; and Videos 5 and 6, available at http:www.jcb.org/cgi/content/full/jcb.200406177/DC1) extends these studies and shows that rasG cells, compared with wild-type cells, exhibit reduced cell polarity and directionality (persistent movement toward the chemoattractant source).

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