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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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Myosin-based contractility causes membrane ruffling. (A) Time-lapse montage of the ruffling membranes. HT1080 cells were plated on collagen-coated, glass-bottomed dishes. Membrane dynamics were observed by phase-contrast microscopy. White, red, and green arrows indicate the edges of distinct membrane protrusions. Black double-headed arrows indicate a change in extent of membrane protrusion. The kymograph shown in B was generated along the white line. Scale bar, 10 μm. (B) Kymograph analysis. White broken lines indicate the timing of ruffling of the membrane protrusions. Scale bars, 10 μm (vertical), 5 min (horizontal). (C, D) Myosin II dynamics. HT1080 cells were treated with control (C) or srGAP1 RNAi (D) and then transfected with VENUS-MRLC (green) and Lifeact-mCherry (red). Kymographs were generated along the white line. Time-lapse images of the area indicated by the white square are shown. Scale bars, 10 μm (white), 10 μm (vertical, kymograph), and 5 min (horizontal, kymograph). Arrows and arrowheads in C indicate the retracting edge of the membrane protrusions. Arrows and arrowheads in D and E indicate actin bundles crossing through lamellipodia.
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Figure 7: Myosin-based contractility causes membrane ruffling. (A) Time-lapse montage of the ruffling membranes. HT1080 cells were plated on collagen-coated, glass-bottomed dishes. Membrane dynamics were observed by phase-contrast microscopy. White, red, and green arrows indicate the edges of distinct membrane protrusions. Black double-headed arrows indicate a change in extent of membrane protrusion. The kymograph shown in B was generated along the white line. Scale bar, 10 μm. (B) Kymograph analysis. White broken lines indicate the timing of ruffling of the membrane protrusions. Scale bars, 10 μm (vertical), 5 min (horizontal). (C, D) Myosin II dynamics. HT1080 cells were treated with control (C) or srGAP1 RNAi (D) and then transfected with VENUS-MRLC (green) and Lifeact-mCherry (red). Kymographs were generated along the white line. Time-lapse images of the area indicated by the white square are shown. Scale bars, 10 μm (white), 10 μm (vertical, kymograph), and 5 min (horizontal, kymograph). Arrows and arrowheads in C indicate the retracting edge of the membrane protrusions. Arrows and arrowheads in D and E indicate actin bundles crossing through lamellipodia.

Mentions: Analysis by phase-contrast microscopy demonstrated that the membrane began to curl upward from the lateral sides and then the protruding edge retracted toward the cell body (Figure 7A, arrows). These observations predict that the contractile force works not only perpendicular, but also parallel, to the extending edge. When the ruffling membrane stopped retracting, the next protruding membrane began to curl upward (Figure 7B, white broken lines). These observations suggest that the contractile force generated at the retracting membrane induces the next extending membrane to curl upward.


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

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

Myosin-based contractility causes membrane ruffling. (A) Time-lapse montage of the ruffling membranes. HT1080 cells were plated on collagen-coated, glass-bottomed dishes. Membrane dynamics were observed by phase-contrast microscopy. White, red, and green arrows indicate the edges of distinct membrane protrusions. Black double-headed arrows indicate a change in extent of membrane protrusion. The kymograph shown in B was generated along the white line. Scale bar, 10 μm. (B) Kymograph analysis. White broken lines indicate the timing of ruffling of the membrane protrusions. Scale bars, 10 μm (vertical), 5 min (horizontal). (C, D) Myosin II dynamics. HT1080 cells were treated with control (C) or srGAP1 RNAi (D) and then transfected with VENUS-MRLC (green) and Lifeact-mCherry (red). Kymographs were generated along the white line. Time-lapse images of the area indicated by the white square are shown. Scale bars, 10 μm (white), 10 μm (vertical, kymograph), and 5 min (horizontal, kymograph). Arrows and arrowheads in C indicate the retracting edge of the membrane protrusions. Arrows and arrowheads in D and E indicate actin bundles crossing through lamellipodia.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814155&req=5

Figure 7: Myosin-based contractility causes membrane ruffling. (A) Time-lapse montage of the ruffling membranes. HT1080 cells were plated on collagen-coated, glass-bottomed dishes. Membrane dynamics were observed by phase-contrast microscopy. White, red, and green arrows indicate the edges of distinct membrane protrusions. Black double-headed arrows indicate a change in extent of membrane protrusion. The kymograph shown in B was generated along the white line. Scale bar, 10 μm. (B) Kymograph analysis. White broken lines indicate the timing of ruffling of the membrane protrusions. Scale bars, 10 μm (vertical), 5 min (horizontal). (C, D) Myosin II dynamics. HT1080 cells were treated with control (C) or srGAP1 RNAi (D) and then transfected with VENUS-MRLC (green) and Lifeact-mCherry (red). Kymographs were generated along the white line. Time-lapse images of the area indicated by the white square are shown. Scale bars, 10 μm (white), 10 μm (vertical, kymograph), and 5 min (horizontal, kymograph). Arrows and arrowheads in C indicate the retracting edge of the membrane protrusions. Arrows and arrowheads in D and E indicate actin bundles crossing through lamellipodia.
Mentions: Analysis by phase-contrast microscopy demonstrated that the membrane began to curl upward from the lateral sides and then the protruding edge retracted toward the cell body (Figure 7A, arrows). These observations predict that the contractile force works not only perpendicular, but also parallel, to the extending edge. When the ruffling membrane stopped retracting, the next protruding membrane began to curl upward (Figure 7B, white broken lines). These observations suggest that the contractile force generated at the retracting membrane induces the next extending membrane to curl upward.

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