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PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms.

Buonomo R, Giacco F, Vasaturo A, Caserta S, Guido S, Pagliara V, Garbi C, Mansueto G, Cassese A, Perruolo G, Oriente F, Miele C, Beguinot F, Formisano P - J. Cell. Physiol. (2012)

Bottom Line: Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts.These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques.At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2).

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology and Pathology, Federico II University of Naples, Naples, Italy.

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Cytoplasmic spreading and cytoskeleton organization in Wt and TgPED fibroblasts. A: Fibroblasts from Wt and TgPED mice were subjected to spreading assays. After 3 h plating on fibronectin, cytoplasmic spreading was quantified by light microscope after staining with crystal violet as described in “Materials and methods” Section. Bars represent the mean ± SD of three independent determinations in triplicate. Asterisks denote statistically significant differences (**P < 0.01). B: Immunofluorescence analysis was performed as described in “Materials and methods” Section. Focal adhesion plaques formation were investigated by immunostaining with specific anti-paxillin antibody. Stress fibers organization were detected by rhodaminate–phalloidin staining. The experiment was repeated four times with similar results. Representative images are shown. The scale bar is 15 µm.
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fig03: Cytoplasmic spreading and cytoskeleton organization in Wt and TgPED fibroblasts. A: Fibroblasts from Wt and TgPED mice were subjected to spreading assays. After 3 h plating on fibronectin, cytoplasmic spreading was quantified by light microscope after staining with crystal violet as described in “Materials and methods” Section. Bars represent the mean ± SD of three independent determinations in triplicate. Asterisks denote statistically significant differences (**P < 0.01). B: Immunofluorescence analysis was performed as described in “Materials and methods” Section. Focal adhesion plaques formation were investigated by immunostaining with specific anti-paxillin antibody. Stress fibers organization were detected by rhodaminate–phalloidin staining. The experiment was repeated four times with similar results. Representative images are shown. The scale bar is 15 µm.

Mentions: Fibroblast motility is dependent on cell ability to adhere to substrates and to transfer into the cytoplasm the signal that induces the cytoskeleton reorganization (Ridley et al., 2003; Arnaout et al., 2007). TgPED fibroblasts showed no significant difference in adhesion compared to the controls (data not shown). Moreover, after 3 h plating on fibronectin, about 60% of Wt cells exhibited a spread cytoplasm. By contrast, the number of spread cells was about 30% for TgPED (Fig. 3A). To analyze actin cytoskeleton and organization of focal adhesion plaques, Wt and TgPED fibroblasts were stained with rhodamin-conjugated phalloidin or with specific anti-paxillin antibodies, respectively. Clearly, the results showed that at least 70% of TgPED fibroblasts displayed a marked decrease of stress fibers and focal adhesion plaques compared to controls (Fig. 3B).


PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms.

Buonomo R, Giacco F, Vasaturo A, Caserta S, Guido S, Pagliara V, Garbi C, Mansueto G, Cassese A, Perruolo G, Oriente F, Miele C, Beguinot F, Formisano P - J. Cell. Physiol. (2012)

Cytoplasmic spreading and cytoskeleton organization in Wt and TgPED fibroblasts. A: Fibroblasts from Wt and TgPED mice were subjected to spreading assays. After 3 h plating on fibronectin, cytoplasmic spreading was quantified by light microscope after staining with crystal violet as described in “Materials and methods” Section. Bars represent the mean ± SD of three independent determinations in triplicate. Asterisks denote statistically significant differences (**P < 0.01). B: Immunofluorescence analysis was performed as described in “Materials and methods” Section. Focal adhesion plaques formation were investigated by immunostaining with specific anti-paxillin antibody. Stress fibers organization were detected by rhodaminate–phalloidin staining. The experiment was repeated four times with similar results. Representative images are shown. The scale bar is 15 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306794&req=5

fig03: Cytoplasmic spreading and cytoskeleton organization in Wt and TgPED fibroblasts. A: Fibroblasts from Wt and TgPED mice were subjected to spreading assays. After 3 h plating on fibronectin, cytoplasmic spreading was quantified by light microscope after staining with crystal violet as described in “Materials and methods” Section. Bars represent the mean ± SD of three independent determinations in triplicate. Asterisks denote statistically significant differences (**P < 0.01). B: Immunofluorescence analysis was performed as described in “Materials and methods” Section. Focal adhesion plaques formation were investigated by immunostaining with specific anti-paxillin antibody. Stress fibers organization were detected by rhodaminate–phalloidin staining. The experiment was repeated four times with similar results. Representative images are shown. The scale bar is 15 µm.
Mentions: Fibroblast motility is dependent on cell ability to adhere to substrates and to transfer into the cytoplasm the signal that induces the cytoskeleton reorganization (Ridley et al., 2003; Arnaout et al., 2007). TgPED fibroblasts showed no significant difference in adhesion compared to the controls (data not shown). Moreover, after 3 h plating on fibronectin, about 60% of Wt cells exhibited a spread cytoplasm. By contrast, the number of spread cells was about 30% for TgPED (Fig. 3A). To analyze actin cytoskeleton and organization of focal adhesion plaques, Wt and TgPED fibroblasts were stained with rhodamin-conjugated phalloidin or with specific anti-paxillin antibodies, respectively. Clearly, the results showed that at least 70% of TgPED fibroblasts displayed a marked decrease of stress fibers and focal adhesion plaques compared to controls (Fig. 3B).

Bottom Line: Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts.These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques.At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2).

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology and Pathology, Federico II University of Naples, Naples, Italy.

Show MeSH
Related in: MedlinePlus