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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Migration of primary MEFs from Ajuba  mice is inhibited. (A) Phase-contrast images of scratch-wound assays of WT and Ajuba  (−/−) MEFs over a 10-h time course. (B) Stick figure of Ajuba and the NH2-terminal PreLIM region and COOH-terminal LIM region. Percent wound closure at 12 h of wt MEFs (+/+, column 1) or Ajuba  MEFs (−/−, columns 2–6) transfected with: RFP alone (+RFP), full-length Ajuba (+RFP-Ajuba), the PreLIM region of Ajuba (+RFP-PreLIM), the LIM region of Ajuba (+RFP-LIM), or both PreLIM and LIM regions of Ajuba expressed by separate plasmids (+GFP-PreLIM and RFP-LIM). WT cells are arbitrarily set at 100%. Multiple wounds were examined and data presented as the mean value plus the SD about the mean.
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fig2: Migration of primary MEFs from Ajuba mice is inhibited. (A) Phase-contrast images of scratch-wound assays of WT and Ajuba (−/−) MEFs over a 10-h time course. (B) Stick figure of Ajuba and the NH2-terminal PreLIM region and COOH-terminal LIM region. Percent wound closure at 12 h of wt MEFs (+/+, column 1) or Ajuba MEFs (−/−, columns 2–6) transfected with: RFP alone (+RFP), full-length Ajuba (+RFP-Ajuba), the PreLIM region of Ajuba (+RFP-PreLIM), the LIM region of Ajuba (+RFP-LIM), or both PreLIM and LIM regions of Ajuba expressed by separate plasmids (+GFP-PreLIM and RFP-LIM). WT cells are arbitrarily set at 100%. Multiple wounds were examined and data presented as the mean value plus the SD about the mean.

Mentions: To determine whether Ajuba played a role in cell migration, early passage MEFs from Ajuba embryos and wt littermates were analyzed. Scratch-wound repair assays of MEFs in culture were performed. Ajuba MEFs displayed a marked inhibition of wound repair compared with litter-matched wt MEFs (Fig. 2 A). By 10 h after wounding, wt MEFs had virtually closed the wound, whereas MEFs from Ajuba mice had only migrated to cover 20–30% of the wound area (Fig. 2 A). By 24 h, Ajuba MEFs closed the wound, indicating that migration of Ajuba cells was inhibited not blocked. In a second approach, classic Boyden chamber migration assays were performed. As in the scratch-wound assay the number of Ajuba MEFs that migrated across the membrane was reduced by 40%, compared with wt MEFs (unpublished data).


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)

Migration of primary MEFs from Ajuba  mice is inhibited. (A) Phase-contrast images of scratch-wound assays of WT and Ajuba  (−/−) MEFs over a 10-h time course. (B) Stick figure of Ajuba and the NH2-terminal PreLIM region and COOH-terminal LIM region. Percent wound closure at 12 h of wt MEFs (+/+, column 1) or Ajuba  MEFs (−/−, columns 2–6) transfected with: RFP alone (+RFP), full-length Ajuba (+RFP-Ajuba), the PreLIM region of Ajuba (+RFP-PreLIM), the LIM region of Ajuba (+RFP-LIM), or both PreLIM and LIM regions of Ajuba expressed by separate plasmids (+GFP-PreLIM and RFP-LIM). WT cells are arbitrarily set at 100%. Multiple wounds were examined and data presented as the mean value plus the SD about the mean.
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Related In: Results  -  Collection

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

fig2: Migration of primary MEFs from Ajuba mice is inhibited. (A) Phase-contrast images of scratch-wound assays of WT and Ajuba (−/−) MEFs over a 10-h time course. (B) Stick figure of Ajuba and the NH2-terminal PreLIM region and COOH-terminal LIM region. Percent wound closure at 12 h of wt MEFs (+/+, column 1) or Ajuba MEFs (−/−, columns 2–6) transfected with: RFP alone (+RFP), full-length Ajuba (+RFP-Ajuba), the PreLIM region of Ajuba (+RFP-PreLIM), the LIM region of Ajuba (+RFP-LIM), or both PreLIM and LIM regions of Ajuba expressed by separate plasmids (+GFP-PreLIM and RFP-LIM). WT cells are arbitrarily set at 100%. Multiple wounds were examined and data presented as the mean value plus the SD about the mean.
Mentions: To determine whether Ajuba played a role in cell migration, early passage MEFs from Ajuba embryos and wt littermates were analyzed. Scratch-wound repair assays of MEFs in culture were performed. Ajuba MEFs displayed a marked inhibition of wound repair compared with litter-matched wt MEFs (Fig. 2 A). By 10 h after wounding, wt MEFs had virtually closed the wound, whereas MEFs from Ajuba mice had only migrated to cover 20–30% of the wound area (Fig. 2 A). By 24 h, Ajuba MEFs closed the wound, indicating that migration of Ajuba cells was inhibited not blocked. In a second approach, classic Boyden chamber migration assays were performed. As in the scratch-wound assay the number of Ajuba MEFs that migrated across the membrane was reduced by 40%, compared with wt MEFs (unpublished data).

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