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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 regulates the cellular migratory behavior. (A) Morphological changes of the motile cells. HT1080 cells treated with control or srGAP1-directed siRNA were plated on collagen-coated, glass-bottomed dishes, and cell migration was monitored by phase-contrast microscopy. White curves indicate the position of the sheet-like membrane protrusions. Arrows indicate the splitting of the protrusions. Scale bar, 100 μm. (B) Quantification of the frequency of splitting of the protrusions. The number of splittings of the membrane protrusions in each cell for 5 h were counted. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars, SEM. ***p < 0.001. (C) Migration tracks of cells plated on collagen-coated substrates. Ten cells from three independent experiments. (D) Quantification of migratory velocity. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. ***p < 0.001. (E) Quantification of the persistence of migratory directionality. D/T is the ratio of the direct distance (D) divided by the total track distance (T). The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, **p < 0.01.
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Figure 9: srGAP1 regulates the cellular migratory behavior. (A) Morphological changes of the motile cells. HT1080 cells treated with control or srGAP1-directed siRNA were plated on collagen-coated, glass-bottomed dishes, and cell migration was monitored by phase-contrast microscopy. White curves indicate the position of the sheet-like membrane protrusions. Arrows indicate the splitting of the protrusions. Scale bar, 100 μm. (B) Quantification of the frequency of splitting of the protrusions. The number of splittings of the membrane protrusions in each cell for 5 h were counted. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars, SEM. ***p < 0.001. (C) Migration tracks of cells plated on collagen-coated substrates. Ten cells from three independent experiments. (D) Quantification of migratory velocity. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. ***p < 0.001. (E) Quantification of the persistence of migratory directionality. D/T is the ratio of the direct distance (D) divided by the total track distance (T). The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, **p < 0.01.

Mentions: To examine the significance of srGAP1 in cell migration, we examined the relation between lamellipodial protrusions and migratory behavior by phase-contrast time-lapse microscopy in control and srGAP1-depleted cells. On collagen-coated substrate, the HT1080 cells showed random migration characterized by repeated directional changes (Figure 9A and Supple­mental Movie S3). When a cell moved in a particular direction, only one compact lamellipodial protrusion was observed at the leading edge. The protrusion was then split into two or three, and the cells stopped moving (Figure 9A, arrows; Andrew and Insall, 2007). When all except one of the membrane protrusions were diminished, the cells began to move in the direction of the left protrusion. Thus cell movement correlates well with the dynamics of lamellipodial protrusions.


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 regulates the cellular migratory behavior. (A) Morphological changes of the motile cells. HT1080 cells treated with control or srGAP1-directed siRNA were plated on collagen-coated, glass-bottomed dishes, and cell migration was monitored by phase-contrast microscopy. White curves indicate the position of the sheet-like membrane protrusions. Arrows indicate the splitting of the protrusions. Scale bar, 100 μm. (B) Quantification of the frequency of splitting of the protrusions. The number of splittings of the membrane protrusions in each cell for 5 h were counted. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars, SEM. ***p < 0.001. (C) Migration tracks of cells plated on collagen-coated substrates. Ten cells from three independent experiments. (D) Quantification of migratory velocity. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. ***p < 0.001. (E) Quantification of the persistence of migratory directionality. D/T is the ratio of the direct distance (D) divided by the total track distance (T). The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, **p < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814155&req=5

Figure 9: srGAP1 regulates the cellular migratory behavior. (A) Morphological changes of the motile cells. HT1080 cells treated with control or srGAP1-directed siRNA were plated on collagen-coated, glass-bottomed dishes, and cell migration was monitored by phase-contrast microscopy. White curves indicate the position of the sheet-like membrane protrusions. Arrows indicate the splitting of the protrusions. Scale bar, 100 μm. (B) Quantification of the frequency of splitting of the protrusions. The number of splittings of the membrane protrusions in each cell for 5 h were counted. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars, SEM. ***p < 0.001. (C) Migration tracks of cells plated on collagen-coated substrates. Ten cells from three independent experiments. (D) Quantification of migratory velocity. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. ***p < 0.001. (E) Quantification of the persistence of migratory directionality. D/T is the ratio of the direct distance (D) divided by the total track distance (T). The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, **p < 0.01.
Mentions: To examine the significance of srGAP1 in cell migration, we examined the relation between lamellipodial protrusions and migratory behavior by phase-contrast time-lapse microscopy in control and srGAP1-depleted cells. On collagen-coated substrate, the HT1080 cells showed random migration characterized by repeated directional changes (Figure 9A and Supple­mental Movie S3). When a cell moved in a particular direction, only one compact lamellipodial protrusion was observed at the leading edge. The protrusion was then split into two or three, and the cells stopped moving (Figure 9A, arrows; Andrew and Insall, 2007). When all except one of the membrane protrusions were diminished, the cells began to move in the direction of the left protrusion. Thus cell movement correlates well with the dynamics of lamellipodial protrusions.

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