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

Imaging of RhoA activity. (A–D) Cells were treated with the indicated siRNAs and then transfected with FRET probes for RhoA. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. White line indicates an outline of an srGAP1-depleted cell. (B) Magnified images of the boxed areas in A. Scale bar, 20 μm. (C, D) Emission ratio of YFP/CFP was measured at the entire region (C) and the membrane protrusions of the cells (D) by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, ***p < 0.001. (E–G) HT1080 cells were transfected with FRET probes for RhoA and the indicated expression vectors. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. (F) The magnified images of the boxed areas in E. Scale bar, 20 μm. (G) Emission ratio of YFP/CFP measured at the membrane protrusions by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. Unpaired Student's t test indicates a significant difference between samples. ****p < 0.0001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814155&req=5

Figure 6: Imaging of RhoA activity. (A–D) Cells were treated with the indicated siRNAs and then transfected with FRET probes for RhoA. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. White line indicates an outline of an srGAP1-depleted cell. (B) Magnified images of the boxed areas in A. Scale bar, 20 μm. (C, D) Emission ratio of YFP/CFP was measured at the entire region (C) and the membrane protrusions of the cells (D) by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, ***p < 0.001. (E–G) HT1080 cells were transfected with FRET probes for RhoA and the indicated expression vectors. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. (F) The magnified images of the boxed areas in E. Scale bar, 20 μm. (G) Emission ratio of YFP/CFP measured at the membrane protrusions by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. Unpaired Student's t test indicates a significant difference between samples. ****p < 0.0001.

Mentions: Previous studies showed that RhoA is activated at the leading edge of the cell (Kurokawa and Matsuda, 2005; Pertz et al., 2006; Machacek et al., 2009), and our study demonstrated that RhoA and Rac1 activities increase toward lamellipodial protrusions in control cells (Figure 6A). In comparison, RhoA activation at lamellipodia was suppressed in srGAP1-depleted cells (Figure 6, A–D). As reported in previous studies (Kurokawa and Matsuda, 2005; Pertz et al., 2006; Wu et al., 2009), expression of Rac1G12V repressed the activation of RhoA in a manner similar to srGAP1 depletion (Figure 6 E–G). Therefore the abolition of RhoA activity may underlie the abnormal lamellipodial dynamics caused by srGAP1 depletion. Indeed, expression of a constitutively active mutant of RhoA (RhoAG14V) recovered membrane ruffling in srGAP1-depleted cells (Supplemental Figure S10).


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

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

Imaging of RhoA activity. (A–D) Cells were treated with the indicated siRNAs and then transfected with FRET probes for RhoA. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. White line indicates an outline of an srGAP1-depleted cell. (B) Magnified images of the boxed areas in A. Scale bar, 20 μm. (C, D) Emission ratio of YFP/CFP was measured at the entire region (C) and the membrane protrusions of the cells (D) by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, ***p < 0.001. (E–G) HT1080 cells were transfected with FRET probes for RhoA and the indicated expression vectors. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. (F) The magnified images of the boxed areas in E. Scale bar, 20 μm. (G) Emission ratio of YFP/CFP measured at the membrane protrusions by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. Unpaired Student's t test indicates a significant difference between samples. ****p < 0.0001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Imaging of RhoA activity. (A–D) Cells were treated with the indicated siRNAs and then transfected with FRET probes for RhoA. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. White line indicates an outline of an srGAP1-depleted cell. (B) Magnified images of the boxed areas in A. Scale bar, 20 μm. (C, D) Emission ratio of YFP/CFP was measured at the entire region (C) and the membrane protrusions of the cells (D) by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. *p < 0.05, ***p < 0.001. (E–G) HT1080 cells were transfected with FRET probes for RhoA and the indicated expression vectors. Cells were imaged for YFP, CFP, and DIC every 20 s for 20 min. FRET efficiencies are shown as YFP/CFP ratio images. (F) The magnified images of the boxed areas in E. Scale bar, 20 μm. (G) Emission ratio of YFP/CFP measured at the membrane protrusions by MetaMorph software. The total number of analyzed cells is shown above each bar. Three independent experiments were performed. Error bars, SEM. Unpaired Student's t test indicates a significant difference between samples. ****p < 0.0001.
Mentions: Previous studies showed that RhoA is activated at the leading edge of the cell (Kurokawa and Matsuda, 2005; Pertz et al., 2006; Machacek et al., 2009), and our study demonstrated that RhoA and Rac1 activities increase toward lamellipodial protrusions in control cells (Figure 6A). In comparison, RhoA activation at lamellipodia was suppressed in srGAP1-depleted cells (Figure 6, A–D). As reported in previous studies (Kurokawa and Matsuda, 2005; Pertz et al., 2006; Wu et al., 2009), expression of Rac1G12V repressed the activation of RhoA in a manner similar to srGAP1 depletion (Figure 6 E–G). Therefore the abolition of RhoA activity may underlie the abnormal lamellipodial dynamics caused by srGAP1 depletion. Indeed, expression of a constitutively active mutant of RhoA (RhoAG14V) recovered membrane ruffling in srGAP1-depleted cells (Supplemental Figure S10).

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