Limits...
The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration.

Pratt SJ, Epple H, Ward M, Feng Y, Braga VM, Longmore GD - J. Cell Biol. (2005)

Bottom Line: Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes.In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis.Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University, St. Louis, MO 63130, USA.

ABSTRACT
Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

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Lamellipodia production by migrating Ajuba  MEFs was abnormal. Phalloidin staining of multiple wt (A) and Ajuba  (−/−) (B) MEFs at the wound edge. The direction of migration is toward the top of the page. Arrows identify ruffles/lamellipodia. Both images were obtained using a 20× lens NA 1.4. (C) Quantitative lamellipodia assay as described in Materials and methods. To control for random lamellipodia production control filters were coated with BSA at each time point. Pseudopodia/lamellipodia production versus time of serum stimulation is presented as protein produced (OD562). Error bars represent the SD from the mean of multiple experiments.
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fig4: Lamellipodia production by migrating Ajuba MEFs was abnormal. Phalloidin staining of multiple wt (A) and Ajuba (−/−) (B) MEFs at the wound edge. The direction of migration is toward the top of the page. Arrows identify ruffles/lamellipodia. Both images were obtained using a 20× lens NA 1.4. (C) Quantitative lamellipodia assay as described in Materials and methods. To control for random lamellipodia production control filters were coated with BSA at each time point. Pseudopodia/lamellipodia production versus time of serum stimulation is presented as protein produced (OD562). Error bars represent the SD from the mean of multiple experiments.

Mentions: A hallmark of migrating cells is the production of lamellipodia at the forward leading edge. To assess the possibility that the migration defect in Ajuba cells could be due to altered lamellipodia production, cells were induced to migrate, by scratch wounding, fixed, and stained with rhodamine-phalloidin to visualize filamentous actin. Migrating wt MEFs at the wound front produced broad lamellipodia and ruffles at their leading edge, as expected (Fig. 4 A, arrows; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1), whereas Ajuba MEFs produced smaller lamellipodia and little ruffling (Fig. 4 B, arrows; Video 3).


The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration.

Pratt SJ, Epple H, Ward M, Feng Y, Braga VM, Longmore GD - J. Cell Biol. (2005)

Lamellipodia production by migrating Ajuba  MEFs was abnormal. Phalloidin staining of multiple wt (A) and Ajuba  (−/−) (B) MEFs at the wound edge. The direction of migration is toward the top of the page. Arrows identify ruffles/lamellipodia. Both images were obtained using a 20× lens NA 1.4. (C) Quantitative lamellipodia assay as described in Materials and methods. To control for random lamellipodia production control filters were coated with BSA at each time point. Pseudopodia/lamellipodia production versus time of serum stimulation is presented as protein produced (OD562). Error bars represent the SD from the mean of multiple experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171823&req=5

fig4: Lamellipodia production by migrating Ajuba MEFs was abnormal. Phalloidin staining of multiple wt (A) and Ajuba (−/−) (B) MEFs at the wound edge. The direction of migration is toward the top of the page. Arrows identify ruffles/lamellipodia. Both images were obtained using a 20× lens NA 1.4. (C) Quantitative lamellipodia assay as described in Materials and methods. To control for random lamellipodia production control filters were coated with BSA at each time point. Pseudopodia/lamellipodia production versus time of serum stimulation is presented as protein produced (OD562). Error bars represent the SD from the mean of multiple experiments.
Mentions: A hallmark of migrating cells is the production of lamellipodia at the forward leading edge. To assess the possibility that the migration defect in Ajuba cells could be due to altered lamellipodia production, cells were induced to migrate, by scratch wounding, fixed, and stained with rhodamine-phalloidin to visualize filamentous actin. Migrating wt MEFs at the wound front produced broad lamellipodia and ruffles at their leading edge, as expected (Fig. 4 A, arrows; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1), whereas Ajuba MEFs produced smaller lamellipodia and little ruffling (Fig. 4 B, arrows; Video 3).

Bottom Line: Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes.In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis.Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University, St. Louis, MO 63130, USA.

ABSTRACT
Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

Show MeSH
Related in: MedlinePlus