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.

Show MeSH

Related in: MedlinePlus

Migration induced Rac activation is reduced in Ajuba  MEFs. (A) WT (+/+, lanes 1, 3, 5–8) or Ajuba  (−/−, lanes 2, 4, 9–12) MEFs were starved overnight (lanes 1 and 2), stimulated with PDGF (lanes 3 and 4) or scratch wounded for the indicated times (lanes 5–12). Rac Western blots for GTP-bound Rac pull downs (top) and total Rac (bottom). (B) Rac-FRET analysis of migrating wt (+/+) and Ajuba  (−/−) MEFs. Cells are migrating toward the top of the page. Red and blue indicate high and low FRET activity, respectively. Scales for each image are shown to the right. (C) Quantification of FRET activity. Average FRET/CFP pixel intensity of the leading edge versus cell body in wt (+/+) versus Ajuba  (−/−) MEFs are shown. 30 or more cells were analyzed for each sample.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171823&req=5

fig6: Migration induced Rac activation is reduced in Ajuba MEFs. (A) WT (+/+, lanes 1, 3, 5–8) or Ajuba (−/−, lanes 2, 4, 9–12) MEFs were starved overnight (lanes 1 and 2), stimulated with PDGF (lanes 3 and 4) or scratch wounded for the indicated times (lanes 5–12). Rac Western blots for GTP-bound Rac pull downs (top) and total Rac (bottom). (B) Rac-FRET analysis of migrating wt (+/+) and Ajuba (−/−) MEFs. Cells are migrating toward the top of the page. Red and blue indicate high and low FRET activity, respectively. Scales for each image are shown to the right. (C) Quantification of FRET activity. Average FRET/CFP pixel intensity of the leading edge versus cell body in wt (+/+) versus Ajuba (−/−) MEFs are shown. 30 or more cells were analyzed for each sample.

Mentions: The Rho family of GTPases is a central regulator of cell migration. Rac1 activity, in particular, is important for lamellipodia production, and the formation of new focal complexes at the leading edge. Because Ajuba cells exhibited abnormalities in both processes we asked whether Rac1 activation, in response to migratory cues, was defective in these cells. To determine Rac1 activity in migrating Ajuba cells both biochemical and imaging analyses were performed. First, multiple scratch wounds were made in a confluent plate of cells, and resultant active GTP-bound Rac1 levels in cell lysates determined by pull-down assay (Etienne-Manneville and Hall, 2001). Although PDGF stimulation of starved MEFs resulted in equivalent activation of Rac1 in wt and Ajuba cells (Fig. 6 A), when MEFs were induced to migrate wt cells exhibited a robust activation of Rac1, whereas Rac1 activation in Ajuba MEFs was significantly blunted (Fig. 6 A).


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 induced Rac activation is reduced in Ajuba  MEFs. (A) WT (+/+, lanes 1, 3, 5–8) or Ajuba  (−/−, lanes 2, 4, 9–12) MEFs were starved overnight (lanes 1 and 2), stimulated with PDGF (lanes 3 and 4) or scratch wounded for the indicated times (lanes 5–12). Rac Western blots for GTP-bound Rac pull downs (top) and total Rac (bottom). (B) Rac-FRET analysis of migrating wt (+/+) and Ajuba  (−/−) MEFs. Cells are migrating toward the top of the page. Red and blue indicate high and low FRET activity, respectively. Scales for each image are shown to the right. (C) Quantification of FRET activity. Average FRET/CFP pixel intensity of the leading edge versus cell body in wt (+/+) versus Ajuba  (−/−) MEFs are shown. 30 or more cells were analyzed for each sample.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Migration induced Rac activation is reduced in Ajuba MEFs. (A) WT (+/+, lanes 1, 3, 5–8) or Ajuba (−/−, lanes 2, 4, 9–12) MEFs were starved overnight (lanes 1 and 2), stimulated with PDGF (lanes 3 and 4) or scratch wounded for the indicated times (lanes 5–12). Rac Western blots for GTP-bound Rac pull downs (top) and total Rac (bottom). (B) Rac-FRET analysis of migrating wt (+/+) and Ajuba (−/−) MEFs. Cells are migrating toward the top of the page. Red and blue indicate high and low FRET activity, respectively. Scales for each image are shown to the right. (C) Quantification of FRET activity. Average FRET/CFP pixel intensity of the leading edge versus cell body in wt (+/+) versus Ajuba (−/−) MEFs are shown. 30 or more cells were analyzed for each sample.
Mentions: The Rho family of GTPases is a central regulator of cell migration. Rac1 activity, in particular, is important for lamellipodia production, and the formation of new focal complexes at the leading edge. Because Ajuba cells exhibited abnormalities in both processes we asked whether Rac1 activation, in response to migratory cues, was defective in these cells. To determine Rac1 activity in migrating Ajuba cells both biochemical and imaging analyses were performed. First, multiple scratch wounds were made in a confluent plate of cells, and resultant active GTP-bound Rac1 levels in cell lysates determined by pull-down assay (Etienne-Manneville and Hall, 2001). Although PDGF stimulation of starved MEFs resulted in equivalent activation of Rac1 in wt and Ajuba cells (Fig. 6 A), when MEFs were induced to migrate wt cells exhibited a robust activation of Rac1, whereas Rac1 activation in Ajuba MEFs was significantly blunted (Fig. 6 A).

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