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Human Mena associates with Rac1 small GTPase in glioblastoma cell lines.

Higashi M, Ishikawa C, Yu J, Toyoda A, Kawana H, Kurokawa K, Matsuda M, Kitagawa M, Harigaya K - PLoS ONE (2009)

Bottom Line: Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia.This cellular phenotype is canceled by introduction of a dominant negative form of Rac1.These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Tumor Pathology, Chiba University Graduate School of Medicine, Chuo-ku, Chiba, Japan.

ABSTRACT
Mammarian enabled (Mena), a member of the Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) family of proteins, has been implicated in cell motility through regulation of the actin cytoskeleton assembly, including lamellipodial protrusion. Rac1, a member of the Rho family GTPases, also plays a pivotal role in the formation of lamellipodia. Here we report that human Mena (hMena) colocalizes with Rac1 in lamellipodia, and using an unmixing assisted acceptor depletion fluorescence resonance energy transfer (u-adFRET) analysis that hMena associates with Rac1 in vivo in the glioblastoma cell line U251MG. Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia. This cellular phenotype is canceled by introduction of a dominant negative form of Rac1. A Rac activity assay and FRET analysis showed that hMena knock-down cells increased the activation of Rac1 at the lamellipodia. These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1.

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Dominant negative Rac1, but not dominant negative RhoA, cancel the increased cell spreading caused by depletion of hMena expression.(A–D) U251MG cells were spread and had vast lamellipodia (B). Introduction of the dominant negative form of Rac1 (C) inhibited the increased spread caused by the introduction of hMena siRNA640 to levels comparable with cells transfected with control siRNA (A). Introduction of the dominant negative form of RhoA GTPase did not inhibit the effect of hMena siRNA (D). Bars, 20 µm. (E). Protein expression of dominant negative Rac1, dominant negative RhoA (upper panel) and hMena (lower panel). (F–G). Box and whisker plot for relative cell area (F), relative perimeter (G) and percent lamellipodia of perimeter (H). Top and bottom of the box represent the 75th and 25th quartile, and whiskers 10th and 90th percentiles, respectively. The middle line of the box is the median. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001). n.s. indicates not significant.
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pone-0004765-g005: Dominant negative Rac1, but not dominant negative RhoA, cancel the increased cell spreading caused by depletion of hMena expression.(A–D) U251MG cells were spread and had vast lamellipodia (B). Introduction of the dominant negative form of Rac1 (C) inhibited the increased spread caused by the introduction of hMena siRNA640 to levels comparable with cells transfected with control siRNA (A). Introduction of the dominant negative form of RhoA GTPase did not inhibit the effect of hMena siRNA (D). Bars, 20 µm. (E). Protein expression of dominant negative Rac1, dominant negative RhoA (upper panel) and hMena (lower panel). (F–G). Box and whisker plot for relative cell area (F), relative perimeter (G) and percent lamellipodia of perimeter (H). Top and bottom of the box represent the 75th and 25th quartile, and whiskers 10th and 90th percentiles, respectively. The middle line of the box is the median. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001). n.s. indicates not significant.

Mentions: Rac is known to play important roles in cell spreading, lamellipodia formation and cell migration [32]. Cell shape changes shown in Figures 3 and 4 were highly reminiscent of the lamellipodial outgrowth caused by activation of Rac. In addition, recent reports demonstrated significantly enhanced activation of the Rac/p21-activated kinase pathway in VASP −/− cells [13]. Therefore, we speculated that knock-down of hMena elicits intracellular signals to activate Rac, and this causes the membrane ruffling and the lamellipodial protrusion. To confirm this hypothesis, we examined whether a dominant negative form of Rac1 (N17Rac1) can cancel the effect caused by introducing siRNA against hMena. U251MG cells were co-transfected with hMena siRNA and CFP tagged-N17Rac1 or a CFP tagged dominant negative form of RhoA (N19RhoA), and subjected to fluorescent microscopy. The expression level of CFP-N17Rac1 and CFP-N19RhoA were similar from Western blot analysis (Figure 5E). Consistent with the previous report, CFP-N17Rac localized at the cell periphery and in the cytoplasm, and CFP-N19RhoA existed diffusely in the cytoplasm [33]. Transfection with CFP or CFP-N19RhoA did not reduce the area or the perimeter of the knock-down cells (Figure 5, A, B, and D). CFP-N17Rac1 inhibited the increased spread caused by the introduction of hMena siRNA to levels comparable with cells transfected with control siRNA (Figure 5C). Statistical analysis revealed that cells transfected with CFP-N17Rac, but not with CFP or CFP-N19RhoA, reduced the cell area, perimeter, and percent lamellipodia of the perimeter to the levels of the control cells (Figure 5, F–H, Table 4). These results indicated that Rac1 is a key molecule involved in the morphological change caused by depletion of hMena.


Human Mena associates with Rac1 small GTPase in glioblastoma cell lines.

Higashi M, Ishikawa C, Yu J, Toyoda A, Kawana H, Kurokawa K, Matsuda M, Kitagawa M, Harigaya K - PLoS ONE (2009)

Dominant negative Rac1, but not dominant negative RhoA, cancel the increased cell spreading caused by depletion of hMena expression.(A–D) U251MG cells were spread and had vast lamellipodia (B). Introduction of the dominant negative form of Rac1 (C) inhibited the increased spread caused by the introduction of hMena siRNA640 to levels comparable with cells transfected with control siRNA (A). Introduction of the dominant negative form of RhoA GTPase did not inhibit the effect of hMena siRNA (D). Bars, 20 µm. (E). Protein expression of dominant negative Rac1, dominant negative RhoA (upper panel) and hMena (lower panel). (F–G). Box and whisker plot for relative cell area (F), relative perimeter (G) and percent lamellipodia of perimeter (H). Top and bottom of the box represent the 75th and 25th quartile, and whiskers 10th and 90th percentiles, respectively. The middle line of the box is the median. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001). n.s. indicates not significant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651628&req=5

pone-0004765-g005: Dominant negative Rac1, but not dominant negative RhoA, cancel the increased cell spreading caused by depletion of hMena expression.(A–D) U251MG cells were spread and had vast lamellipodia (B). Introduction of the dominant negative form of Rac1 (C) inhibited the increased spread caused by the introduction of hMena siRNA640 to levels comparable with cells transfected with control siRNA (A). Introduction of the dominant negative form of RhoA GTPase did not inhibit the effect of hMena siRNA (D). Bars, 20 µm. (E). Protein expression of dominant negative Rac1, dominant negative RhoA (upper panel) and hMena (lower panel). (F–G). Box and whisker plot for relative cell area (F), relative perimeter (G) and percent lamellipodia of perimeter (H). Top and bottom of the box represent the 75th and 25th quartile, and whiskers 10th and 90th percentiles, respectively. The middle line of the box is the median. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001). n.s. indicates not significant.
Mentions: Rac is known to play important roles in cell spreading, lamellipodia formation and cell migration [32]. Cell shape changes shown in Figures 3 and 4 were highly reminiscent of the lamellipodial outgrowth caused by activation of Rac. In addition, recent reports demonstrated significantly enhanced activation of the Rac/p21-activated kinase pathway in VASP −/− cells [13]. Therefore, we speculated that knock-down of hMena elicits intracellular signals to activate Rac, and this causes the membrane ruffling and the lamellipodial protrusion. To confirm this hypothesis, we examined whether a dominant negative form of Rac1 (N17Rac1) can cancel the effect caused by introducing siRNA against hMena. U251MG cells were co-transfected with hMena siRNA and CFP tagged-N17Rac1 or a CFP tagged dominant negative form of RhoA (N19RhoA), and subjected to fluorescent microscopy. The expression level of CFP-N17Rac1 and CFP-N19RhoA were similar from Western blot analysis (Figure 5E). Consistent with the previous report, CFP-N17Rac localized at the cell periphery and in the cytoplasm, and CFP-N19RhoA existed diffusely in the cytoplasm [33]. Transfection with CFP or CFP-N19RhoA did not reduce the area or the perimeter of the knock-down cells (Figure 5, A, B, and D). CFP-N17Rac1 inhibited the increased spread caused by the introduction of hMena siRNA to levels comparable with cells transfected with control siRNA (Figure 5C). Statistical analysis revealed that cells transfected with CFP-N17Rac, but not with CFP or CFP-N19RhoA, reduced the cell area, perimeter, and percent lamellipodia of the perimeter to the levels of the control cells (Figure 5, F–H, Table 4). These results indicated that Rac1 is a key molecule involved in the morphological change caused by depletion of hMena.

Bottom Line: Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia.This cellular phenotype is canceled by introduction of a dominant negative form of Rac1.These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Tumor Pathology, Chiba University Graduate School of Medicine, Chuo-ku, Chiba, Japan.

ABSTRACT
Mammarian enabled (Mena), a member of the Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) family of proteins, has been implicated in cell motility through regulation of the actin cytoskeleton assembly, including lamellipodial protrusion. Rac1, a member of the Rho family GTPases, also plays a pivotal role in the formation of lamellipodia. Here we report that human Mena (hMena) colocalizes with Rac1 in lamellipodia, and using an unmixing assisted acceptor depletion fluorescence resonance energy transfer (u-adFRET) analysis that hMena associates with Rac1 in vivo in the glioblastoma cell line U251MG. Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia. This cellular phenotype is canceled by introduction of a dominant negative form of Rac1. A Rac activity assay and FRET analysis showed that hMena knock-down cells increased the activation of Rac1 at the lamellipodia. These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1.

Show MeSH
Related in: MedlinePlus