Limits...
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.

Show MeSH

Related in: MedlinePlus

srGAP1 regulates the properties of the sheet-like membrane protrusions. (A) Western blot analysis of HT1080 cells transfected with the indicated siRNAs and the rescue constructs. (B) Cells treated with the indicated siRNAs were stained for srGAP1 (green) and actin filament (F-actin; red). Magnified images of the areas indicated by the white squares. Scale bar, 20 μm. (C) Morphologies of srGAP1-depleted cells. Cells transfected with the indicated siRNAs and the GFP-fused rescue constructs (green) were stained for F-actin (red). Magnified images of the boxed areas indicated by the white squares (left, GFP; middle, F-actin; right, merged). Scale bar, 20 μm. (D) Quantification of the morphologies of sheet-like membrane protrusions. Cells transfected with the indicated siRNAs and rescue constructs were stained for F-actin. Cells were classified on the basis of morphology of the membrane protrusion. Cells more than half of whose protrusive area was ruffling were considered ruffling. Cells more than half of whose protrusive area was spreading were considered spreading. Cells without the sheet-like membrane protrusions were considered no. We analyzed 150 cells from three independent experiments. Error bars indicate SEM. (E) Quantification of the frequency of membrane ruffling. We counted the number of rufflings of the largest membrane protrusions in each cell for 20 min. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (F) Quantification of the perimeter of the sheet-like membrane protrusions. The total number of analyzed protrusions is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (G) Quantification of the number of sheet-like membrane protrusions. Thirty cells were analyzed from three independent experiments. Error bars indicate SEM. **p < 0.01, ***p < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814155&req=5

Figure 1: srGAP1 regulates the properties of the sheet-like membrane protrusions. (A) Western blot analysis of HT1080 cells transfected with the indicated siRNAs and the rescue constructs. (B) Cells treated with the indicated siRNAs were stained for srGAP1 (green) and actin filament (F-actin; red). Magnified images of the areas indicated by the white squares. Scale bar, 20 μm. (C) Morphologies of srGAP1-depleted cells. Cells transfected with the indicated siRNAs and the GFP-fused rescue constructs (green) were stained for F-actin (red). Magnified images of the boxed areas indicated by the white squares (left, GFP; middle, F-actin; right, merged). Scale bar, 20 μm. (D) Quantification of the morphologies of sheet-like membrane protrusions. Cells transfected with the indicated siRNAs and rescue constructs were stained for F-actin. Cells were classified on the basis of morphology of the membrane protrusion. Cells more than half of whose protrusive area was ruffling were considered ruffling. Cells more than half of whose protrusive area was spreading were considered spreading. Cells without the sheet-like membrane protrusions were considered no. We analyzed 150 cells from three independent experiments. Error bars indicate SEM. (E) Quantification of the frequency of membrane ruffling. We counted the number of rufflings of the largest membrane protrusions in each cell for 20 min. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (F) Quantification of the perimeter of the sheet-like membrane protrusions. The total number of analyzed protrusions is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (G) Quantification of the number of sheet-like membrane protrusions. Thirty cells were analyzed from three independent experiments. Error bars indicate SEM. **p < 0.01, ***p < 0.001.

Mentions: Immunostaining revealed that endogenous srGAP1 is localized to the tip of the ruffling membranes in HT1080 fibrosarcoma cells (Figure 1, A and B). To examine the role of srGAP1 in the control of lamellipodia, we repressed its expression by RNA interference (RNAi) in HT1080 cells (Figure 1A). Depletion of srGAP1 did not affect the ratio of cells with lamellipodial protrusions but altered their morphology (Figure 1, C and D). When treated with the control small interfering RNA (siRNA), 60% of cells showed ruffling membranes and 20% had spreading membranes. In the srGAP1-depleted cells, the relative number of cells with ruffling membranes decreased and the number of cells with spreading membranes increased in comparison with control cells (Figure 1D). To analyze such morphological differences in more detail, we observed the dynamics of membrane protrusions by time-lapse imaging using phase-contrast microscopy and analyzed them by kymography, which produces a time line of protrusion and retraction. In control cells with ruffling membranes, membrane extension was interrupted by ruffling of the membrane followed by retraction (Supplemental Figure S1A and Supplemental Movie S1). The cycle, which was composed of ruffling and retraction of the membrane protrusions, was continuously and periodically repeated, thereby resulting in the ruffling structure of the lamellipodia in the HT1080 cells. Depletion of srGAP1 decreased the frequency of membrane ruffling and increased the persistence of membrane extension, which resulted in the spreading structure of lamellipodia with less ruffling (Figure 1E, Supplemental Figure S1B, and Supplemental Movie S2). Thus the spreading morphology of protrusions is due to decreased ruffling in srGAP1-depleted cells.


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 properties of the sheet-like membrane protrusions. (A) Western blot analysis of HT1080 cells transfected with the indicated siRNAs and the rescue constructs. (B) Cells treated with the indicated siRNAs were stained for srGAP1 (green) and actin filament (F-actin; red). Magnified images of the areas indicated by the white squares. Scale bar, 20 μm. (C) Morphologies of srGAP1-depleted cells. Cells transfected with the indicated siRNAs and the GFP-fused rescue constructs (green) were stained for F-actin (red). Magnified images of the boxed areas indicated by the white squares (left, GFP; middle, F-actin; right, merged). Scale bar, 20 μm. (D) Quantification of the morphologies of sheet-like membrane protrusions. Cells transfected with the indicated siRNAs and rescue constructs were stained for F-actin. Cells were classified on the basis of morphology of the membrane protrusion. Cells more than half of whose protrusive area was ruffling were considered ruffling. Cells more than half of whose protrusive area was spreading were considered spreading. Cells without the sheet-like membrane protrusions were considered no. We analyzed 150 cells from three independent experiments. Error bars indicate SEM. (E) Quantification of the frequency of membrane ruffling. We counted the number of rufflings of the largest membrane protrusions in each cell for 20 min. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (F) Quantification of the perimeter of the sheet-like membrane protrusions. The total number of analyzed protrusions is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (G) Quantification of the number of sheet-like membrane protrusions. Thirty cells were analyzed from three independent experiments. Error bars indicate SEM. **p < 0.01, ***p < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: srGAP1 regulates the properties of the sheet-like membrane protrusions. (A) Western blot analysis of HT1080 cells transfected with the indicated siRNAs and the rescue constructs. (B) Cells treated with the indicated siRNAs were stained for srGAP1 (green) and actin filament (F-actin; red). Magnified images of the areas indicated by the white squares. Scale bar, 20 μm. (C) Morphologies of srGAP1-depleted cells. Cells transfected with the indicated siRNAs and the GFP-fused rescue constructs (green) were stained for F-actin (red). Magnified images of the boxed areas indicated by the white squares (left, GFP; middle, F-actin; right, merged). Scale bar, 20 μm. (D) Quantification of the morphologies of sheet-like membrane protrusions. Cells transfected with the indicated siRNAs and rescue constructs were stained for F-actin. Cells were classified on the basis of morphology of the membrane protrusion. Cells more than half of whose protrusive area was ruffling were considered ruffling. Cells more than half of whose protrusive area was spreading were considered spreading. Cells without the sheet-like membrane protrusions were considered no. We analyzed 150 cells from three independent experiments. Error bars indicate SEM. (E) Quantification of the frequency of membrane ruffling. We counted the number of rufflings of the largest membrane protrusions in each cell for 20 min. Three independent experiments were performed. The total number of analyzed cells is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (F) Quantification of the perimeter of the sheet-like membrane protrusions. The total number of analyzed protrusions is shown above each bar. Error bars indicate SEM. **p < 0.01, ***p < 0.001. (G) Quantification of the number of sheet-like membrane protrusions. Thirty cells were analyzed from three independent experiments. Error bars indicate SEM. **p < 0.01, ***p < 0.001.
Mentions: Immunostaining revealed that endogenous srGAP1 is localized to the tip of the ruffling membranes in HT1080 fibrosarcoma cells (Figure 1, A and B). To examine the role of srGAP1 in the control of lamellipodia, we repressed its expression by RNA interference (RNAi) in HT1080 cells (Figure 1A). Depletion of srGAP1 did not affect the ratio of cells with lamellipodial protrusions but altered their morphology (Figure 1, C and D). When treated with the control small interfering RNA (siRNA), 60% of cells showed ruffling membranes and 20% had spreading membranes. In the srGAP1-depleted cells, the relative number of cells with ruffling membranes decreased and the number of cells with spreading membranes increased in comparison with control cells (Figure 1D). To analyze such morphological differences in more detail, we observed the dynamics of membrane protrusions by time-lapse imaging using phase-contrast microscopy and analyzed them by kymography, which produces a time line of protrusion and retraction. In control cells with ruffling membranes, membrane extension was interrupted by ruffling of the membrane followed by retraction (Supplemental Figure S1A and Supplemental Movie S1). The cycle, which was composed of ruffling and retraction of the membrane protrusions, was continuously and periodically repeated, thereby resulting in the ruffling structure of the lamellipodia in the HT1080 cells. Depletion of srGAP1 decreased the frequency of membrane ruffling and increased the persistence of membrane extension, which resulted in the spreading structure of lamellipodia with less ruffling (Figure 1E, Supplemental Figure S1B, and Supplemental Movie S2). Thus the spreading morphology of protrusions is due to decreased ruffling in srGAP1-depleted cells.

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