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The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

West KA, Zhang H, Brown MC, Nikolopoulos SN, Riedy MC, Horwitz AF, Turner CE - J. Cell Biol. (2001)

Bottom Line: In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif.Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells.These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA.

ABSTRACT
The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

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Overexpression of paxillinΔLD4 affects the protrusive activity and motility of cells. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells were plated on fibronectin (2 μg/ml), and motility was analyzed by time-lapse video microscopy. PaxillinΔLD4 cells were much more dynamic than paxillin WT and parental nontransfected cells, extending multiple lamellipodia (arrows). Long retraction fiber-like extensions were also observed (arrowhead). (B) Quantification of protrusiveness demonstrates an increase in cell area (in μm2) of paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (C) Quantification of random motility on fibronectin (2 μg/ml) demonstrates an increase in locomotion (in μm/hr) in paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (D) Quantification of random motility to fibronectin (10 μg/ml) reveals a striking increase in the percentage of paxillinΔLD4 cells migrating to immobilized fibronectin, as compared with paxillin WT, paxillinΔLD2, and parental nontransfected cells. Values are the average of modified Boyden chamber assays performed in quadruplicate. The migration of parental nontransfected CHO.K1 cells was set to 100%, and the other cell types were measured against this value. (E) Scrape wound assays using paxillinΔLD4 and paxillin WT cells plated in 35-mm tissue culture dishes in complete media at a confluent density and scored with a micropipet tip demonstrate that, although paxillin WT cells are able close the wound, paxillinΔLD4 are severely retarded in this capacity. The images are representative of experiments performed in triplicate.
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fig3: Overexpression of paxillinΔLD4 affects the protrusive activity and motility of cells. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells were plated on fibronectin (2 μg/ml), and motility was analyzed by time-lapse video microscopy. PaxillinΔLD4 cells were much more dynamic than paxillin WT and parental nontransfected cells, extending multiple lamellipodia (arrows). Long retraction fiber-like extensions were also observed (arrowhead). (B) Quantification of protrusiveness demonstrates an increase in cell area (in μm2) of paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (C) Quantification of random motility on fibronectin (2 μg/ml) demonstrates an increase in locomotion (in μm/hr) in paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (D) Quantification of random motility to fibronectin (10 μg/ml) reveals a striking increase in the percentage of paxillinΔLD4 cells migrating to immobilized fibronectin, as compared with paxillin WT, paxillinΔLD2, and parental nontransfected cells. Values are the average of modified Boyden chamber assays performed in quadruplicate. The migration of parental nontransfected CHO.K1 cells was set to 100%, and the other cell types were measured against this value. (E) Scrape wound assays using paxillinΔLD4 and paxillin WT cells plated in 35-mm tissue culture dishes in complete media at a confluent density and scored with a micropipet tip demonstrate that, although paxillin WT cells are able close the wound, paxillinΔLD4 are severely retarded in this capacity. The images are representative of experiments performed in triplicate.

Mentions: The formation of membrane ruffles and lamellipodia are characteristics of motile cells (Mitchison and Cramer, 1996). To test a role for paxillin's LD4 motif in the regulation of cell motility, paxillinΔLD4, paxillin WT and parental nontransfected CHO.K1 cells were plated on fibronectin and then analyzed using time-lapse video microscopy. Interestingly, paxillinΔLD4 cells were highly active, extending multiple lamellipodia (Fig. 3 A, arrows, and B). In addition, long retraction fiber-like processes were observed (Fig. 3 A, arrowheads). In contrast, paxillin WT and parental nontransfected cells displayed little protrusive activity (Fig. 3, A and B). The exaggerated protrusive activity of the paxillinΔLD4 cells was accompanied by a significant increase in the rate of random motility of these cells over paxillin WT and parental nontransfected cell populations (Fig. 3 C). To test the effects of the deletion of paxillin LD4 motif on haptotaxis, modified Boyden chamber migration assays were performed. In support of the time-lapse video microscopy data, paxillinΔLD4 cells migrated at a higher rate than both paxillin WT and parental nontransfected cells (Fig. 3 D). Interestingly, overexpressing paxillin constructs engineered with a deletion of the second LD motif (paxillinΔLD2) in CHO.K1 cells did not affect migration (Fig. 3 D). Focal adhesion kinase (FAK), a protein implicated in cell motility, has been shown to associate with paxillin through both LD2 and LD4 (Turner and Miller, 1994; Ilic et al., 1995; Brown et al., 1996; Cary et al., 1996; Turner et al., 1999). This suggests that the increases in random motility caused by deletion of the LD4 motif were not due to the uncoupling of FAK and paxillin.


The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

West KA, Zhang H, Brown MC, Nikolopoulos SN, Riedy MC, Horwitz AF, Turner CE - J. Cell Biol. (2001)

Overexpression of paxillinΔLD4 affects the protrusive activity and motility of cells. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells were plated on fibronectin (2 μg/ml), and motility was analyzed by time-lapse video microscopy. PaxillinΔLD4 cells were much more dynamic than paxillin WT and parental nontransfected cells, extending multiple lamellipodia (arrows). Long retraction fiber-like extensions were also observed (arrowhead). (B) Quantification of protrusiveness demonstrates an increase in cell area (in μm2) of paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (C) Quantification of random motility on fibronectin (2 μg/ml) demonstrates an increase in locomotion (in μm/hr) in paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (D) Quantification of random motility to fibronectin (10 μg/ml) reveals a striking increase in the percentage of paxillinΔLD4 cells migrating to immobilized fibronectin, as compared with paxillin WT, paxillinΔLD2, and parental nontransfected cells. Values are the average of modified Boyden chamber assays performed in quadruplicate. The migration of parental nontransfected CHO.K1 cells was set to 100%, and the other cell types were measured against this value. (E) Scrape wound assays using paxillinΔLD4 and paxillin WT cells plated in 35-mm tissue culture dishes in complete media at a confluent density and scored with a micropipet tip demonstrate that, although paxillin WT cells are able close the wound, paxillinΔLD4 are severely retarded in this capacity. The images are representative of experiments performed in triplicate.
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Related In: Results  -  Collection

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fig3: Overexpression of paxillinΔLD4 affects the protrusive activity and motility of cells. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells were plated on fibronectin (2 μg/ml), and motility was analyzed by time-lapse video microscopy. PaxillinΔLD4 cells were much more dynamic than paxillin WT and parental nontransfected cells, extending multiple lamellipodia (arrows). Long retraction fiber-like extensions were also observed (arrowhead). (B) Quantification of protrusiveness demonstrates an increase in cell area (in μm2) of paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (C) Quantification of random motility on fibronectin (2 μg/ml) demonstrates an increase in locomotion (in μm/hr) in paxillinΔLD4 cells, as compared with paxillin WT and parental nontransfected cells. (D) Quantification of random motility to fibronectin (10 μg/ml) reveals a striking increase in the percentage of paxillinΔLD4 cells migrating to immobilized fibronectin, as compared with paxillin WT, paxillinΔLD2, and parental nontransfected cells. Values are the average of modified Boyden chamber assays performed in quadruplicate. The migration of parental nontransfected CHO.K1 cells was set to 100%, and the other cell types were measured against this value. (E) Scrape wound assays using paxillinΔLD4 and paxillin WT cells plated in 35-mm tissue culture dishes in complete media at a confluent density and scored with a micropipet tip demonstrate that, although paxillin WT cells are able close the wound, paxillinΔLD4 are severely retarded in this capacity. The images are representative of experiments performed in triplicate.
Mentions: The formation of membrane ruffles and lamellipodia are characteristics of motile cells (Mitchison and Cramer, 1996). To test a role for paxillin's LD4 motif in the regulation of cell motility, paxillinΔLD4, paxillin WT and parental nontransfected CHO.K1 cells were plated on fibronectin and then analyzed using time-lapse video microscopy. Interestingly, paxillinΔLD4 cells were highly active, extending multiple lamellipodia (Fig. 3 A, arrows, and B). In addition, long retraction fiber-like processes were observed (Fig. 3 A, arrowheads). In contrast, paxillin WT and parental nontransfected cells displayed little protrusive activity (Fig. 3, A and B). The exaggerated protrusive activity of the paxillinΔLD4 cells was accompanied by a significant increase in the rate of random motility of these cells over paxillin WT and parental nontransfected cell populations (Fig. 3 C). To test the effects of the deletion of paxillin LD4 motif on haptotaxis, modified Boyden chamber migration assays were performed. In support of the time-lapse video microscopy data, paxillinΔLD4 cells migrated at a higher rate than both paxillin WT and parental nontransfected cells (Fig. 3 D). Interestingly, overexpressing paxillin constructs engineered with a deletion of the second LD motif (paxillinΔLD2) in CHO.K1 cells did not affect migration (Fig. 3 D). Focal adhesion kinase (FAK), a protein implicated in cell motility, has been shown to associate with paxillin through both LD2 and LD4 (Turner and Miller, 1994; Ilic et al., 1995; Brown et al., 1996; Cary et al., 1996; Turner et al., 1999). This suggests that the increases in random motility caused by deletion of the LD4 motif were not due to the uncoupling of FAK and paxillin.

Bottom Line: In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif.Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells.These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA.

ABSTRACT
The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

Show MeSH
Related in: MedlinePlus