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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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Characterization of the F-BAR–FX unit of srGAP1. (A) GST-fusion proteins were incubated with or without brain liposomes and then centrifuged. Supernatant (S) and pellet (P) were separated by SDS–PAGE. Proteins were detected by Coomassie brilliant blue. Arrowheads indicate a bacteria-derived protein copurified during affinity purification of GST-fusion proteins. (B) FLAG-Rac1G12V and FLAG-Rac1T17N were expressed in COS7 cells and then subjected to pull-down assay with GST-F-BAR and GST-CT, respectively. The bound proteins were analyzed by immunoblotting with anti-FLAG antibody. Arrows indicate GST-fusion proteins. (C) FLAG-Rac1G12V and myc-srGAP1 (F-BAR, FX, and F-BAR–FX) were expressed in COS7 cells and then immunoprecipitated by anti-FLAG antibody. The bound proteins were analyzed by immunoblotting with anti- FLAG antibody. A bracket indicates FLAG-F-BAR–FX. (D) Bacterially expressed Rac1 was loaded with GTPγS (GTPgS) and GDP and subjected to pull-down assay with GST-F-BAR. The bound proteins were analyzed by immunoblotting with anti-Rac antibody.
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Figure 5: Characterization of the F-BAR–FX unit of srGAP1. (A) GST-fusion proteins were incubated with or without brain liposomes and then centrifuged. Supernatant (S) and pellet (P) were separated by SDS–PAGE. Proteins were detected by Coomassie brilliant blue. Arrowheads indicate a bacteria-derived protein copurified during affinity purification of GST-fusion proteins. (B) FLAG-Rac1G12V and FLAG-Rac1T17N were expressed in COS7 cells and then subjected to pull-down assay with GST-F-BAR and GST-CT, respectively. The bound proteins were analyzed by immunoblotting with anti-FLAG antibody. Arrows indicate GST-fusion proteins. (C) FLAG-Rac1G12V and myc-srGAP1 (F-BAR, FX, and F-BAR–FX) were expressed in COS7 cells and then immunoprecipitated by anti-FLAG antibody. The bound proteins were analyzed by immunoblotting with anti- FLAG antibody. A bracket indicates FLAG-F-BAR–FX. (D) Bacterially expressed Rac1 was loaded with GTPγS (GTPgS) and GDP and subjected to pull-down assay with GST-F-BAR. The bound proteins were analyzed by immunoblotting with anti-Rac antibody.

Mentions: The BAR domain, which is structurally similar to the F-BAR domain, binds with both acidic phospholipids and small GTPases (Habermann, 2004; de Kreuk et al., 2011). Indeed, each of the F-BAR and FX domains of srGAP1 possesses lipid-binding activity (Figure 5A). The F-BAR domain of srGAP1 directly interacts with Rac1, and the affinity between both proteins increases in a manner dependent on Rac1 activity (Figure 5, B–D), suggesting the importance of F-BAR–FX unit and Rac1 interactions in Rac1-induced membrane localization of srGAP1. Because the CT region also bound with Rac1 (Figure 5B), such interaction might contribute Rac1-induced recruitment of srGAP1 to the membrane.


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

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

Characterization of the F-BAR–FX unit of srGAP1. (A) GST-fusion proteins were incubated with or without brain liposomes and then centrifuged. Supernatant (S) and pellet (P) were separated by SDS–PAGE. Proteins were detected by Coomassie brilliant blue. Arrowheads indicate a bacteria-derived protein copurified during affinity purification of GST-fusion proteins. (B) FLAG-Rac1G12V and FLAG-Rac1T17N were expressed in COS7 cells and then subjected to pull-down assay with GST-F-BAR and GST-CT, respectively. The bound proteins were analyzed by immunoblotting with anti-FLAG antibody. Arrows indicate GST-fusion proteins. (C) FLAG-Rac1G12V and myc-srGAP1 (F-BAR, FX, and F-BAR–FX) were expressed in COS7 cells and then immunoprecipitated by anti-FLAG antibody. The bound proteins were analyzed by immunoblotting with anti- FLAG antibody. A bracket indicates FLAG-F-BAR–FX. (D) Bacterially expressed Rac1 was loaded with GTPγS (GTPgS) and GDP and subjected to pull-down assay with GST-F-BAR. The bound proteins were analyzed by immunoblotting with anti-Rac antibody.
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Related In: Results  -  Collection

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Figure 5: Characterization of the F-BAR–FX unit of srGAP1. (A) GST-fusion proteins were incubated with or without brain liposomes and then centrifuged. Supernatant (S) and pellet (P) were separated by SDS–PAGE. Proteins were detected by Coomassie brilliant blue. Arrowheads indicate a bacteria-derived protein copurified during affinity purification of GST-fusion proteins. (B) FLAG-Rac1G12V and FLAG-Rac1T17N were expressed in COS7 cells and then subjected to pull-down assay with GST-F-BAR and GST-CT, respectively. The bound proteins were analyzed by immunoblotting with anti-FLAG antibody. Arrows indicate GST-fusion proteins. (C) FLAG-Rac1G12V and myc-srGAP1 (F-BAR, FX, and F-BAR–FX) were expressed in COS7 cells and then immunoprecipitated by anti-FLAG antibody. The bound proteins were analyzed by immunoblotting with anti- FLAG antibody. A bracket indicates FLAG-F-BAR–FX. (D) Bacterially expressed Rac1 was loaded with GTPγS (GTPgS) and GDP and subjected to pull-down assay with GST-F-BAR. The bound proteins were analyzed by immunoblotting with anti-Rac antibody.
Mentions: The BAR domain, which is structurally similar to the F-BAR domain, binds with both acidic phospholipids and small GTPases (Habermann, 2004; de Kreuk et al., 2011). Indeed, each of the F-BAR and FX domains of srGAP1 possesses lipid-binding activity (Figure 5A). The F-BAR domain of srGAP1 directly interacts with Rac1, and the affinity between both proteins increases in a manner dependent on Rac1 activity (Figure 5, B–D), suggesting the importance of F-BAR–FX unit and Rac1 interactions in Rac1-induced membrane localization of srGAP1. Because the CT region also bound with Rac1 (Figure 5B), such interaction might contribute Rac1-induced recruitment of srGAP1 to the membrane.

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