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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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Distribution of mCherry-hMena and CFP-wtRac1 in a live cell.After co-transfection of mCherry-hMena and CFP-wtRac1 in U251MG cells, images were obtained every thirty seconds for fifteen minutes with a confocal microscope. mCherry-hMena and CFP-wtRac1 were colocalized in lamellipodia and the cytosol (A–C; also available as Supplemental Movie S1). (D) Intensity correlation analysis. The PDM plot showed a high codependency of hMena and Rac1 distribution in lamellipodia and a moderate codependency in the cytosol. (E) Kymograph of the PDM plot at the line indicated in Figure 1D. (F) Time sequence of the PDM value at the lines indicated in Figure 1E. The PDM value in the lamellipodia (red line) exhibited oscillatory changes, although the PDM value in the cytoplasm (blue line) was comparatively constant.
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pone-0004765-g001: Distribution of mCherry-hMena and CFP-wtRac1 in a live cell.After co-transfection of mCherry-hMena and CFP-wtRac1 in U251MG cells, images were obtained every thirty seconds for fifteen minutes with a confocal microscope. mCherry-hMena and CFP-wtRac1 were colocalized in lamellipodia and the cytosol (A–C; also available as Supplemental Movie S1). (D) Intensity correlation analysis. The PDM plot showed a high codependency of hMena and Rac1 distribution in lamellipodia and a moderate codependency in the cytosol. (E) Kymograph of the PDM plot at the line indicated in Figure 1D. (F) Time sequence of the PDM value at the lines indicated in Figure 1E. The PDM value in the lamellipodia (red line) exhibited oscillatory changes, although the PDM value in the cytoplasm (blue line) was comparatively constant.

Mentions: A time-lapse colocalization analysis between hMena and Rac1 in U251MG cells was examined in order to detect the in vivo interaction and to determine the physical significance of the subcellular localization of hMena and Rac1. Images were obtained every thirty seconds for fifteen minutes using a confocal microscope. Similar to former reports using fibroblasts [4], hMena was distributed at the focal adhesion (Figure S1) and at the edge of the lamellipodia (Figures 1A and S1). CFP-Rac1 localized broadly at the membrane protrusion (Figure 1B). The merged images indicated that hMena and Rac colocalized at the lamellipodia (Figure 1C, Movie S1). As a visual inspection of these images suggested colocalization of the proteins, we used intensity correlation analysis (ICA) [25] to test for a relationship between hMena and Rac1. A pseudocoloured image, where each pixel is equal to the PDM (product from the differences from the means; see Materials and Methods) value at that location (Figure. 1D, Movie S2), showed a high codependency of hMena and Rac1 in the lamellipodia and a moderate codependency in the cytosol. A kymograph of the PDM plot at the line indicated in Figure 1D showed that the PDM value at the ruffling membrane is higher than in the cytosol (Figures 1, E and F, Movie S2).


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)

Distribution of mCherry-hMena and CFP-wtRac1 in a live cell.After co-transfection of mCherry-hMena and CFP-wtRac1 in U251MG cells, images were obtained every thirty seconds for fifteen minutes with a confocal microscope. mCherry-hMena and CFP-wtRac1 were colocalized in lamellipodia and the cytosol (A–C; also available as Supplemental Movie S1). (D) Intensity correlation analysis. The PDM plot showed a high codependency of hMena and Rac1 distribution in lamellipodia and a moderate codependency in the cytosol. (E) Kymograph of the PDM plot at the line indicated in Figure 1D. (F) Time sequence of the PDM value at the lines indicated in Figure 1E. The PDM value in the lamellipodia (red line) exhibited oscillatory changes, although the PDM value in the cytoplasm (blue line) was comparatively constant.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004765-g001: Distribution of mCherry-hMena and CFP-wtRac1 in a live cell.After co-transfection of mCherry-hMena and CFP-wtRac1 in U251MG cells, images were obtained every thirty seconds for fifteen minutes with a confocal microscope. mCherry-hMena and CFP-wtRac1 were colocalized in lamellipodia and the cytosol (A–C; also available as Supplemental Movie S1). (D) Intensity correlation analysis. The PDM plot showed a high codependency of hMena and Rac1 distribution in lamellipodia and a moderate codependency in the cytosol. (E) Kymograph of the PDM plot at the line indicated in Figure 1D. (F) Time sequence of the PDM value at the lines indicated in Figure 1E. The PDM value in the lamellipodia (red line) exhibited oscillatory changes, although the PDM value in the cytoplasm (blue line) was comparatively constant.
Mentions: A time-lapse colocalization analysis between hMena and Rac1 in U251MG cells was examined in order to detect the in vivo interaction and to determine the physical significance of the subcellular localization of hMena and Rac1. Images were obtained every thirty seconds for fifteen minutes using a confocal microscope. Similar to former reports using fibroblasts [4], hMena was distributed at the focal adhesion (Figure S1) and at the edge of the lamellipodia (Figures 1A and S1). CFP-Rac1 localized broadly at the membrane protrusion (Figure 1B). The merged images indicated that hMena and Rac colocalized at the lamellipodia (Figure 1C, Movie S1). As a visual inspection of these images suggested colocalization of the proteins, we used intensity correlation analysis (ICA) [25] to test for a relationship between hMena and Rac1. A pseudocoloured image, where each pixel is equal to the PDM (product from the differences from the means; see Materials and Methods) value at that location (Figure. 1D, Movie S2), showed a high codependency of hMena and Rac1 in the lamellipodia and a moderate codependency in the cytosol. A kymograph of the PDM plot at the line indicated in Figure 1D showed that the PDM value at the ruffling membrane is higher than in the cytosol (Figures 1, E and F, Movie S2).

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