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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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srGAP1 is recruited to the membrane protrusions through its F-BAR–FX unit, dependent on Rac1 signaling. (A) The effect of Rac1 signaling on the recruitment of srGAP1 to the membrane. Myc-srGAP1 (full length and F-BAR–FX) and mCherry-Rac1 (G12V and T17N) were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown (top, myc; middle, F-actin; bottom, merged). Scale bar, 20 μm. (B) Quantification. The signal intensities of myc-srGAP1 (full length and F-BAR–FX) at the tip of the membrane protrusions were measured by ImageJ. The signal intensities at the tip of the membrane were normalized to those at the region 1 μm behind the tip. From three independent experiments, 45 protrusions of 15 cells were analyzed. Error bars indicate SEM. ***p < 0.001. (C) Constructs of srGAP1. (D) Myc-srGAP1 (full length, ∆(F-BAR-FX), F-BAR, FX, F-BAR–FX, ∆GAP, ∆SH3, and ∆CT) and GFP-Rac1G12V were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown. Scale bar, 20 μm. (E) Quantification. Cells in which myc-srGAP1 is localized at the tip of lamellipodial protrusions were counted. From three independent experiments, 150 cells were analyzed. Error bars, SEM.
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Figure 4: srGAP1 is recruited to the membrane protrusions through its F-BAR–FX unit, dependent on Rac1 signaling. (A) The effect of Rac1 signaling on the recruitment of srGAP1 to the membrane. Myc-srGAP1 (full length and F-BAR–FX) and mCherry-Rac1 (G12V and T17N) were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown (top, myc; middle, F-actin; bottom, merged). Scale bar, 20 μm. (B) Quantification. The signal intensities of myc-srGAP1 (full length and F-BAR–FX) at the tip of the membrane protrusions were measured by ImageJ. The signal intensities at the tip of the membrane were normalized to those at the region 1 μm behind the tip. From three independent experiments, 45 protrusions of 15 cells were analyzed. Error bars indicate SEM. ***p < 0.001. (C) Constructs of srGAP1. (D) Myc-srGAP1 (full length, ∆(F-BAR-FX), F-BAR, FX, F-BAR–FX, ∆GAP, ∆SH3, and ∆CT) and GFP-Rac1G12V were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown. Scale bar, 20 μm. (E) Quantification. Cells in which myc-srGAP1 is localized at the tip of lamellipodial protrusions were counted. From three independent experiments, 150 cells were analyzed. Error bars, SEM.

Mentions: To examine the spatial regulation of srGAP1, we ectopically expressed it and observed its cellular localization in COS7 cells. Exogenous srGAP1 showed cytosolic distribution, but the GAP-dead and GAP-deleted mutants (R542A and ∆GAP, respectively) of srGAP1 were localized to the tip of the membrane protrusions (Supplemental Figure S7). These results suggest that GAP-dependent inactivation of endogenous Rac disrupts the membrane localization of srGAP1. Of importance, Rac1G12V expression dramatically increased membrane localization of srGAP1 (Figure 4, A and B). Thus Rac1 signaling is important for membrane recruitment of srGAP1.


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

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

srGAP1 is recruited to the membrane protrusions through its F-BAR–FX unit, dependent on Rac1 signaling. (A) The effect of Rac1 signaling on the recruitment of srGAP1 to the membrane. Myc-srGAP1 (full length and F-BAR–FX) and mCherry-Rac1 (G12V and T17N) were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown (top, myc; middle, F-actin; bottom, merged). Scale bar, 20 μm. (B) Quantification. The signal intensities of myc-srGAP1 (full length and F-BAR–FX) at the tip of the membrane protrusions were measured by ImageJ. The signal intensities at the tip of the membrane were normalized to those at the region 1 μm behind the tip. From three independent experiments, 45 protrusions of 15 cells were analyzed. Error bars indicate SEM. ***p < 0.001. (C) Constructs of srGAP1. (D) Myc-srGAP1 (full length, ∆(F-BAR-FX), F-BAR, FX, F-BAR–FX, ∆GAP, ∆SH3, and ∆CT) and GFP-Rac1G12V were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown. Scale bar, 20 μm. (E) Quantification. Cells in which myc-srGAP1 is localized at the tip of lamellipodial protrusions were counted. From three independent experiments, 150 cells were analyzed. Error bars, SEM.
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Figure 4: srGAP1 is recruited to the membrane protrusions through its F-BAR–FX unit, dependent on Rac1 signaling. (A) The effect of Rac1 signaling on the recruitment of srGAP1 to the membrane. Myc-srGAP1 (full length and F-BAR–FX) and mCherry-Rac1 (G12V and T17N) were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown (top, myc; middle, F-actin; bottom, merged). Scale bar, 20 μm. (B) Quantification. The signal intensities of myc-srGAP1 (full length and F-BAR–FX) at the tip of the membrane protrusions were measured by ImageJ. The signal intensities at the tip of the membrane were normalized to those at the region 1 μm behind the tip. From three independent experiments, 45 protrusions of 15 cells were analyzed. Error bars indicate SEM. ***p < 0.001. (C) Constructs of srGAP1. (D) Myc-srGAP1 (full length, ∆(F-BAR-FX), F-BAR, FX, F-BAR–FX, ∆GAP, ∆SH3, and ∆CT) and GFP-Rac1G12V were coexpressed in COS7 cells. Cells were fixed and then stained for myc-srGAP1 (green) and F-actin (blue). The magnified images of the boxed areas are shown. Scale bar, 20 μm. (E) Quantification. Cells in which myc-srGAP1 is localized at the tip of lamellipodial protrusions were counted. From three independent experiments, 150 cells were analyzed. Error bars, SEM.
Mentions: To examine the spatial regulation of srGAP1, we ectopically expressed it and observed its cellular localization in COS7 cells. Exogenous srGAP1 showed cytosolic distribution, but the GAP-dead and GAP-deleted mutants (R542A and ∆GAP, respectively) of srGAP1 were localized to the tip of the membrane protrusions (Supplemental Figure S7). These results suggest that GAP-dependent inactivation of endogenous Rac disrupts the membrane localization of srGAP1. Of importance, Rac1G12V expression dramatically increased membrane localization of srGAP1 (Figure 4, A and B). Thus Rac1 signaling is important for membrane recruitment of srGAP1.

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