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1.
FIGURE 7.

FIGURE 7. From: LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization.

A summary of the effects of expression or knockdown of LIMK1 or cofilin on the rates of actin polymerization, actin retrograde flow, and lamellipodium extension. A, actin dynamics and width of stationary lamellipodia in RacV12-expressing N1E-115 cells are shown. B, actin dynamics and lamellipodium extension in extending and stationary lamellipodia in NRG-stimulated MCF-7 cells are shown. Details are described in “Discussion.”

Kazumasa Ohashi, et al. J Biol Chem. 2011 October 21;286(42):36340-36351.
2.
FIGURE 1.

FIGURE 1. From: LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization.

Time-lapse fluorescence images of lamellipodia in N1E-115 cells. A, fluorescence images of YFP-actin in an N1E-115 cell cultured in low serum (5%) are shown. B, fluorescence images of YFP-actin in a RacV12-expressing N1E-115 cell are shown. The right image is a magnified view of the boxed region in the left panel. Lp, lamellipodium; Lm, lamella; TB, transverse bundles. In A and B, fluorescence images of YFP-actin were acquired every 30 s for 15 min (see supplemental Movie S1). Scale bars, 10 μm.

Kazumasa Ohashi, et al. J Biol Chem. 2011 October 21;286(42):36340-36351.
3.
FIGURE 4.

FIGURE 4. From: LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization.

FRAP time-lapse measurements of the rates of lamellipodium extension, actin retrograde flow, and actin polymerization in NRG-stimulated MCF-7 cells. A, FRAP analysis of YFP-actin in the lamellipodium of a NRG-stimulated MCF-7 cell is shown. MCF-7 cells transfected with YFP-actin were cultured for 18 h, serum-starved for 5 h, and then stimulated with 50 ng/ml NRG. After photobleaching of a 4.5 × 22.5-μm rectangular region (white box), fluorescence images were acquired every 1 s for 38 s (see supplemental Movie S5). The dashed line indicates the position of the initial cell margin. Scale bar, 10 μm. B, shown is a kymograph analysis for determining the rates of lamellipodium extension, actin retrograde flow, and actin polymerization. The kymograph was obtained by compiling the fluorescence images of a white-lined region (perpendicular to the cell margin) in A. The rates of lamellipodium extension, actin retrograde flow, and actin polymerization were measured as described under “Experimental Procedures.”

Kazumasa Ohashi, et al. J Biol Chem. 2011 October 21;286(42):36340-36351.
4.
FIGURE 6.

FIGURE 6. From: LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization.

Effect of LIMK1 or cofilin knockdown on the rates of actin polymerization, retrograde flow, and lamellipodium extension in NRG-stimulated MCF-7 cells. A, a FRAP time-lapse analysis is shown. MCF-7 cells were cotransfected with YFP-actin and control shRNA, LIMK1 shRNA#1, or cofilin shRNA#1. Cells were cultured for 48 h, treated with NRG, and subjected to FRAP analysis, as in Fig. 5A. See also supplemental Movie S9. Scale bar, 10 μm. B, histograms of cell numbers for lamellipodium extension rates are shown. Lamellipodia were classified into stationary and extension phases, as in Fig. 5B. C, shown is a quantitative analysis of the rates of actin polymerization, retrograde flow, and lamellipodium extension. Lamellipodia were classified into extension and stationary phases, as in B. Data are the means ± S.D. of 21 (control shRNA), 38 (LIMK1 shRNA#1), and 30 cells (cofilin shRNA#1) for extending lamellipodia, and 29 (Control shRNA), 48 (LIMK1 shRNA#1), and 32 cells (Cofilin shRNA#1) for stationary lamellipodia. D, ratios of conversion of actin polymerization into actin retrograde flow and lamellipodium extension are shown. Data are calculated from C. *, p < 0.05; **, p < 0.001.

Kazumasa Ohashi, et al. J Biol Chem. 2011 October 21;286(42):36340-36351.
5.
FIGURE 3.

FIGURE 3. From: LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization.

Effect of knockdown of LIMK1 or cofilin on the rate of actin retrograde flow and the width of lamellipodia in RacV12-expressing N1E-115 cells. A, suppression of LIMK1 or cofilin expression by shRNA plasmids is shown. N1E-115 cells were transfected with control, LIMK1, or cofilin shRNAs. After 48 h of culture, cell lysates were analyzed by immunoblotting with antibodies specific for LIMK1, cofilin, and β-actin. B, shown is a FRAP time-lapse analysis of the effect of LIMK1 or cofilin knockdown on the rate of actin retrograde flow in RacV12-expressing N1E-115 cells. FRAP time-lapse imaging of YFP-actin and kymograph analysis were carried out as in Fig. 2A (see supplemental Movie S3). Scale bar, 5 μm. C, shown is quantification of the rate of actin retrograde flow. D, shown is quantification of the width of lamellipodia. Data are the means ± S.D. of 143 (Control shRNA), 118 (LIMK1 shRNA#1), and 92 cells (Cofilin shRNA#1) from at least three independent experiments. *, p < 0.05.

Kazumasa Ohashi, et al. J Biol Chem. 2011 October 21;286(42):36340-36351.
6.
FIGURE 2.

FIGURE 2. From: LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization.

Effect of expression of LIMK1 or cofilin on the rate of actin retrograde flow and on the width of lamellipodia in RacV12-expressing N1E-115 cells. A, FRAP time-lapse imaging of YFP-actin in RacV12-expressing N1E-115 cells is shown. Cells were cotransfected with CFP (Control), LIMK1-CFP, LIMK1-CFP + cofilin (S3A)-DsRed, LIMK1-CFP + SSH1-CFP, cofilin-CFP, or cofilin (S3A)-CFP. After photobleaching of a 4.5 × 22.5-μm rectangular region (dotted box), fluorescence images were acquired every 1 s for 38 s using a laser-scanning confocal microscopy (see supplemental Movie S2). Scale bar, 5 μm. Panels on the far right show the kymographs of the white lined region (perpendicular to the cell margin) depicted in the far left panels. The positions of the initial cell margin and lamella are indicated by black and white triangles, respectively. B, shown is the rate of actin retrograde flow in lamellipodia, measured by kymograph analysis. C, shown is quantification of the width of lamellipodia. Data in B and C are the means ± S.D. of 142 (control), 81 (LIMK1), 41 (LIMK1 + cofilin (S3A)), 36 (LIMK1 + SSH1), 26 (cofilin), and 41 cells (cofilin (S3A)) from at least three independent experiments. *, p < 0.01; **, p < 0.0001.

Kazumasa Ohashi, et al. J Biol Chem. 2011 October 21;286(42):36340-36351.
7.
FIGURE 5.

FIGURE 5. From: LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization.

Effect of cofilin (S3A) or LIMK1 expression on the rates of actin polymerization, actin retrograde flow, and lamellipodium extension in NRG-stimulated MCF-7 cells. A, FRAP time-lapse analysis is shown. MCF-7 cells were cotransfected with YFP-actin and cofilin (S3A)-CFP or LIMK1-CFP. After serum starvation, cells were treated with NRG and subjected to FRAP analysis, as in Fig. 4A. After photobleaching, fluorescence images were acquired every 1 s for 28 s (see supplemental Movie S8). Kymograph analysis was conducted as in Fig. 4B. The positions of the initial cell margin and lamella are indicated by black and white triangles, respectively. Scale bar, 10 μm. B, shown are histograms of cell numbers for lamellipodium extension rates. Lamellipodia with extension rates lower than 3 μm/min were classified as stationary phase, and lamellipodia with extension rates greater than 3 μm/min were classified as extension phase. C, shown is a quantitative analysis of the rates of actin polymerization, retrograde flow, and lamellipodium extension. Lamellipodia were classified into extension and stationary phases, as in B. Data are the means ± S.D. of 45 (control) and 53 cells (cofilin (S3A)) for extending lamellipodia and 39 (control), 24 (cofilin (S3A)), and 46 cells (LIMK1) for stationary lamellipodia. *, p < 0.005. D, ratios of conversion of actin polymerization into actin retrograde flow and lamellipodium extension are shown. Data are calculated from C. *, p < 0.05.

Kazumasa Ohashi, et al. J Biol Chem. 2011 October 21;286(42):36340-36351.

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