Limits...
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.

Show MeSH

Related in: MedlinePlus

Imaging of Rac activity in U251MG cells using a u-adFRET assay.U251MG cells co-transfected with Raichu-Rac and hMena siRNA or control siRNA were replated onto glass-bottom dishes. YFP and CFP images were obtained from spectral images using the linear unmixing method. Expression patterns of YFP (acceptor) and CFP (donor) of Raichu-Rac1 before (A, B, left panels) and after (A, B, right panels) acceptor photobleaching. Bleached area indicated with a rectangle in A. In the control cells, the whole area in B is photobleached. (A', B') Mapping of FRET efficiency. In hMena knock-down cells, Rac1 was activated in an extended arc-like structure which corresponds to the lamellipodia (A'). In control cells, Rac1 was also activated at the protrusive ruffling membrane (B'). Bars, 20 µm. (C) Example profiles of fluorescence of donor (blue) and acceptor (red) before and after photobleaching. Dashed lines indicate the change of the fluorescence within the non-bleached area. (D) Box and whisker plots for mean FRET efficiency of U251MG cells. (E) Box and whisker plots for regional FRET efficiency in U251MG cells. For the box and whisker plots, 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. Data is from 20 cells each. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2651628&req=5

pone-0004765-g007: Imaging of Rac activity in U251MG cells using a u-adFRET assay.U251MG cells co-transfected with Raichu-Rac and hMena siRNA or control siRNA were replated onto glass-bottom dishes. YFP and CFP images were obtained from spectral images using the linear unmixing method. Expression patterns of YFP (acceptor) and CFP (donor) of Raichu-Rac1 before (A, B, left panels) and after (A, B, right panels) acceptor photobleaching. Bleached area indicated with a rectangle in A. In the control cells, the whole area in B is photobleached. (A', B') Mapping of FRET efficiency. In hMena knock-down cells, Rac1 was activated in an extended arc-like structure which corresponds to the lamellipodia (A'). In control cells, Rac1 was also activated at the protrusive ruffling membrane (B'). Bars, 20 µm. (C) Example profiles of fluorescence of donor (blue) and acceptor (red) before and after photobleaching. Dashed lines indicate the change of the fluorescence within the non-bleached area. (D) Box and whisker plots for mean FRET efficiency of U251MG cells. (E) Box and whisker plots for regional FRET efficiency in U251MG cells. For the box and whisker plots, 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. Data is from 20 cells each. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001).

Mentions: Next, we visualized the activity of Rac1 in cells to obtain direct information about the spatial changes of the activity of Rac1 using a FRET-based in vivo probe. As the in vivo probe, we used a single-molecule pRaichu-Rac1 which consisted of Rac1, the CRIB domain of PAK1, YFP and CFP. Upon activation of Rac1, the binding to PAK-CRIB increases the efficiency of FRET between CFP and YFP [19]. U251MG cells co-transfected with Raichu-Rac1 and siRNA were fixed and analyzed with a quantitative u-adFRET [29], [30]. A similar phenotype of cell spread with the formation of lamellipodia in hMena knock-down cells was also observed in the cells with the introduction of pRaichu-Rac1 probe. Figure 7C shows the time sequence of the mean fluorescence of the donors and acceptors in the region of interest (ROI). After acceptor photobleaching, donor emission within the ROI in Figure 7A (rectangular area) was increased (Figure 7C, solid blue line), while the donor emission within the non-bleached area was not changed (Figure 7C, dashed blue line), indicating that FRET occurred in the ROI shown in Figure 7A. Maps of E calculated from u-adFRET showed that Rac1 was activated at the lamellipodia in either the control or the knock-down cells (Figure 7, A', B' and E). The observations of the activation of Rac1 at the protrusive ruffling membrane are similar to previous reports where different kinds of FRET probes were used [16], [17], [18], [19]. The regional analysis within a cell showed that the E values in the lamellipodia are similar in the control and knock-down cells. The average E value in a whole cell was increased in knock-down cells (Figure 7D).


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)

Imaging of Rac activity in U251MG cells using a u-adFRET assay.U251MG cells co-transfected with Raichu-Rac and hMena siRNA or control siRNA were replated onto glass-bottom dishes. YFP and CFP images were obtained from spectral images using the linear unmixing method. Expression patterns of YFP (acceptor) and CFP (donor) of Raichu-Rac1 before (A, B, left panels) and after (A, B, right panels) acceptor photobleaching. Bleached area indicated with a rectangle in A. In the control cells, the whole area in B is photobleached. (A', B') Mapping of FRET efficiency. In hMena knock-down cells, Rac1 was activated in an extended arc-like structure which corresponds to the lamellipodia (A'). In control cells, Rac1 was also activated at the protrusive ruffling membrane (B'). Bars, 20 µm. (C) Example profiles of fluorescence of donor (blue) and acceptor (red) before and after photobleaching. Dashed lines indicate the change of the fluorescence within the non-bleached area. (D) Box and whisker plots for mean FRET efficiency of U251MG cells. (E) Box and whisker plots for regional FRET efficiency in U251MG cells. For the box and whisker plots, 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. Data is from 20 cells each. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004765-g007: Imaging of Rac activity in U251MG cells using a u-adFRET assay.U251MG cells co-transfected with Raichu-Rac and hMena siRNA or control siRNA were replated onto glass-bottom dishes. YFP and CFP images were obtained from spectral images using the linear unmixing method. Expression patterns of YFP (acceptor) and CFP (donor) of Raichu-Rac1 before (A, B, left panels) and after (A, B, right panels) acceptor photobleaching. Bleached area indicated with a rectangle in A. In the control cells, the whole area in B is photobleached. (A', B') Mapping of FRET efficiency. In hMena knock-down cells, Rac1 was activated in an extended arc-like structure which corresponds to the lamellipodia (A'). In control cells, Rac1 was also activated at the protrusive ruffling membrane (B'). Bars, 20 µm. (C) Example profiles of fluorescence of donor (blue) and acceptor (red) before and after photobleaching. Dashed lines indicate the change of the fluorescence within the non-bleached area. (D) Box and whisker plots for mean FRET efficiency of U251MG cells. (E) Box and whisker plots for regional FRET efficiency in U251MG cells. For the box and whisker plots, 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. Data is from 20 cells each. Brackets with asterisks indicate statistically significant differences between data sets from a Student's t test (p<0.001).
Mentions: Next, we visualized the activity of Rac1 in cells to obtain direct information about the spatial changes of the activity of Rac1 using a FRET-based in vivo probe. As the in vivo probe, we used a single-molecule pRaichu-Rac1 which consisted of Rac1, the CRIB domain of PAK1, YFP and CFP. Upon activation of Rac1, the binding to PAK-CRIB increases the efficiency of FRET between CFP and YFP [19]. U251MG cells co-transfected with Raichu-Rac1 and siRNA were fixed and analyzed with a quantitative u-adFRET [29], [30]. A similar phenotype of cell spread with the formation of lamellipodia in hMena knock-down cells was also observed in the cells with the introduction of pRaichu-Rac1 probe. Figure 7C shows the time sequence of the mean fluorescence of the donors and acceptors in the region of interest (ROI). After acceptor photobleaching, donor emission within the ROI in Figure 7A (rectangular area) was increased (Figure 7C, solid blue line), while the donor emission within the non-bleached area was not changed (Figure 7C, dashed blue line), indicating that FRET occurred in the ROI shown in Figure 7A. Maps of E calculated from u-adFRET showed that Rac1 was activated at the lamellipodia in either the control or the knock-down cells (Figure 7, A', B' and E). The observations of the activation of Rac1 at the protrusive ruffling membrane are similar to previous reports where different kinds of FRET probes were used [16], [17], [18], [19]. The regional analysis within a cell showed that the E values in the lamellipodia are similar in the control and knock-down cells. The average E value in a whole cell was increased in knock-down cells (Figure 7D).

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