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srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity.

Yamazaki D, Itoh T, Miki H, Takenawa T - Mol. Biol. Cell (2013)

Bottom Line: When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility.Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent.Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan Laboratory of Lipid Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

ABSTRACT
The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

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Actomyosin contractile bundles at lamellipodia. (A) HT1080 cells transfected with the indicated siRNAs and the mCherry-tagged rescue constructs (red) were stained for phosphorylated MRLC (S19) (green) and F-actin (blue). Arrowheads indicate the actomyosin contractile bundles. Scale bar, 10 μm. (B) Quantification. Cells with actomyosin bundles at the base of the sheet-like membrane protrusions were counted. One hundred fifty cells were analyzed from three independent experiments. Error bars, SEM. ***p < 0.001.
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Figure 8: Actomyosin contractile bundles at lamellipodia. (A) HT1080 cells transfected with the indicated siRNAs and the mCherry-tagged rescue constructs (red) were stained for phosphorylated MRLC (S19) (green) and F-actin (blue). Arrowheads indicate the actomyosin contractile bundles. Scale bar, 10 μm. (B) Quantification. Cells with actomyosin bundles at the base of the sheet-like membrane protrusions were counted. One hundred fifty cells were analyzed from three independent experiments. Error bars, SEM. ***p < 0.001.

Mentions: In the srGAP1-depleted cells, myosin II showed a spot-like distribution, and colocalization of actin and myosin II was not observed at the spreading lamellipodia (Figure 7D). Although actin bundles were observed in lamellipodial protrusions, they were not covered with myosin II (Figure 7D, arrows and arrowheads). Thus the interaction between actin and myosin II and the formation of actomyosin bundles at the lamellipodia did not occur in srGAP1-depleted cells. The defective formation of actomyosin bundles at the base of the lamellipodia in srGAP1-depleted cells was recovered by reexpression of srGAP1 but not srGAP1R542A (Figure 8, A and B). Furthermore, constitutive activation of Rac1 also inhibited actomyosin bundle formation, whereas loss of Rac1 promoted MRLC phosphorylation and development of cortical actomyosin bundles and membrane blebs dependent on ROCK (Figure 8B and Supplemental Figure S15). Of importance, treatment with blebbistatin or Y27632 also altered the distribution of myosin II and inhibited bundle formation at the lamellipodia (Figure 8B and Supplemental Figures S13, control siRNA, and S16). Collectively these results suggest that srGAP1 regulates Rho/ROCK signaling–mediated actomyosin contractility at lamellipodia via modulation of Rac1 activity.


srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity.

Yamazaki D, Itoh T, Miki H, Takenawa T - Mol. Biol. Cell (2013)

Actomyosin contractile bundles at lamellipodia. (A) HT1080 cells transfected with the indicated siRNAs and the mCherry-tagged rescue constructs (red) were stained for phosphorylated MRLC (S19) (green) and F-actin (blue). Arrowheads indicate the actomyosin contractile bundles. Scale bar, 10 μm. (B) Quantification. Cells with actomyosin bundles at the base of the sheet-like membrane protrusions were counted. One hundred fifty cells were analyzed from three independent experiments. Error bars, SEM. ***p < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: Actomyosin contractile bundles at lamellipodia. (A) HT1080 cells transfected with the indicated siRNAs and the mCherry-tagged rescue constructs (red) were stained for phosphorylated MRLC (S19) (green) and F-actin (blue). Arrowheads indicate the actomyosin contractile bundles. Scale bar, 10 μm. (B) Quantification. Cells with actomyosin bundles at the base of the sheet-like membrane protrusions were counted. One hundred fifty cells were analyzed from three independent experiments. Error bars, SEM. ***p < 0.001.
Mentions: In the srGAP1-depleted cells, myosin II showed a spot-like distribution, and colocalization of actin and myosin II was not observed at the spreading lamellipodia (Figure 7D). Although actin bundles were observed in lamellipodial protrusions, they were not covered with myosin II (Figure 7D, arrows and arrowheads). Thus the interaction between actin and myosin II and the formation of actomyosin bundles at the lamellipodia did not occur in srGAP1-depleted cells. The defective formation of actomyosin bundles at the base of the lamellipodia in srGAP1-depleted cells was recovered by reexpression of srGAP1 but not srGAP1R542A (Figure 8, A and B). Furthermore, constitutive activation of Rac1 also inhibited actomyosin bundle formation, whereas loss of Rac1 promoted MRLC phosphorylation and development of cortical actomyosin bundles and membrane blebs dependent on ROCK (Figure 8B and Supplemental Figure S15). Of importance, treatment with blebbistatin or Y27632 also altered the distribution of myosin II and inhibited bundle formation at the lamellipodia (Figure 8B and Supplemental Figures S13, control siRNA, and S16). Collectively these results suggest that srGAP1 regulates Rho/ROCK signaling–mediated actomyosin contractility at lamellipodia via modulation of Rac1 activity.

Bottom Line: When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility.Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent.Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan Laboratory of Lipid Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

ABSTRACT
The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

Show MeSH
Related in: MedlinePlus