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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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Partial depletion of Rac1 recovers membrane ruffling in srGAP1-depleted cells. (A) Western blot analysis of cells treated with the indicated amounts of Rac1 siRNA. Signal intensity of Rac1 was quantified with ImageJ. The amounts of Rac1 were normalized to those of actin. In the graphs, the amount of Rac1 in cells treated with Rac1 siRNA is normalized to that in nontreated cells. Three independent experiments were performed. Error bars indicate SEM. (B) Cells were treated with control or srGAP1 siRNAs and then with the indicated amounts of Rac1 siRNA after 24 h of the first treatment. After 24 h of the second treatment cells were fixed and stained for F-actin. Bottom, magnified images of the boxed areas in the top. Scale bar, 100 μm (top) and 12.5 μm (bottom). (C) Quantification. Morphologies of the sheet-like membrane protrusions classified as described in Figure 1D. From three independent experiments, 150 cells were analyzed. Error bars indicate SEM.
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Figure 3: Partial depletion of Rac1 recovers membrane ruffling in srGAP1-depleted cells. (A) Western blot analysis of cells treated with the indicated amounts of Rac1 siRNA. Signal intensity of Rac1 was quantified with ImageJ. The amounts of Rac1 were normalized to those of actin. In the graphs, the amount of Rac1 in cells treated with Rac1 siRNA is normalized to that in nontreated cells. Three independent experiments were performed. Error bars indicate SEM. (B) Cells were treated with control or srGAP1 siRNAs and then with the indicated amounts of Rac1 siRNA after 24 h of the first treatment. After 24 h of the second treatment cells were fixed and stained for F-actin. Bottom, magnified images of the boxed areas in the top. Scale bar, 100 μm (top) and 12.5 μm (bottom). (C) Quantification. Morphologies of the sheet-like membrane protrusions classified as described in Figure 1D. From three independent experiments, 150 cells were analyzed. Error bars indicate SEM.

Mentions: To confirm whether changes in the properties of the membrane protrusions in srGAP1-depleted cells were due to increased Rac1 activity, Rac1 we also repressed expression by RNAi (Figure 3A). The partial repression of Rac1 expression by treatment with 0.1 nM Rac1 siRNA recovered membrane ruffling in srGAP1-depleted cells (Figure 3, B and C). Conversely, expression of a constitutively active mutant of Rac1 (Rac1G12V) showed the same effect on lamellipodial protrusions as srGAP1 depletion (Supplemental Figure S5). Furthermore, activation of Rac1 promoted spreading of the protrusions more effectively than that of RhoA and Cdc42 in HT1080 cells (Supplemental Figure S6). These results suggest that srGAP1-dependent regulation of Rac1 activity is important for the control of lamellipodia.


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

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

Partial depletion of Rac1 recovers membrane ruffling in srGAP1-depleted cells. (A) Western blot analysis of cells treated with the indicated amounts of Rac1 siRNA. Signal intensity of Rac1 was quantified with ImageJ. The amounts of Rac1 were normalized to those of actin. In the graphs, the amount of Rac1 in cells treated with Rac1 siRNA is normalized to that in nontreated cells. Three independent experiments were performed. Error bars indicate SEM. (B) Cells were treated with control or srGAP1 siRNAs and then with the indicated amounts of Rac1 siRNA after 24 h of the first treatment. After 24 h of the second treatment cells were fixed and stained for F-actin. Bottom, magnified images of the boxed areas in the top. Scale bar, 100 μm (top) and 12.5 μm (bottom). (C) Quantification. Morphologies of the sheet-like membrane protrusions classified as described in Figure 1D. From three independent experiments, 150 cells were analyzed. Error bars indicate SEM.
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Related In: Results  -  Collection

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Figure 3: Partial depletion of Rac1 recovers membrane ruffling in srGAP1-depleted cells. (A) Western blot analysis of cells treated with the indicated amounts of Rac1 siRNA. Signal intensity of Rac1 was quantified with ImageJ. The amounts of Rac1 were normalized to those of actin. In the graphs, the amount of Rac1 in cells treated with Rac1 siRNA is normalized to that in nontreated cells. Three independent experiments were performed. Error bars indicate SEM. (B) Cells were treated with control or srGAP1 siRNAs and then with the indicated amounts of Rac1 siRNA after 24 h of the first treatment. After 24 h of the second treatment cells were fixed and stained for F-actin. Bottom, magnified images of the boxed areas in the top. Scale bar, 100 μm (top) and 12.5 μm (bottom). (C) Quantification. Morphologies of the sheet-like membrane protrusions classified as described in Figure 1D. From three independent experiments, 150 cells were analyzed. Error bars indicate SEM.
Mentions: To confirm whether changes in the properties of the membrane protrusions in srGAP1-depleted cells were due to increased Rac1 activity, Rac1 we also repressed expression by RNAi (Figure 3A). The partial repression of Rac1 expression by treatment with 0.1 nM Rac1 siRNA recovered membrane ruffling in srGAP1-depleted cells (Figure 3, B and C). Conversely, expression of a constitutively active mutant of Rac1 (Rac1G12V) showed the same effect on lamellipodial protrusions as srGAP1 depletion (Supplemental Figure S5). Furthermore, activation of Rac1 promoted spreading of the protrusions more effectively than that of RhoA and Cdc42 in HT1080 cells (Supplemental Figure S6). These results suggest that srGAP1-dependent regulation of Rac1 activity is important for the control of lamellipodia.

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