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RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages.

von Wichert G, Jiang G, Kostic A, De Vos K, Sap J, Sheetz MP - J. Cell Biol. (2003)

Bottom Line: We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha).RPTPalpha colocalizes with alphav-integrins at the leading edge during early spreading, and coimmunoprecipitates with alphav-integrins during spreading on fibronectin and vitronectin.RPTPalpha-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha). RPTPalpha colocalizes with alphav-integrins at the leading edge during early spreading, and coimmunoprecipitates with alphav-integrins during spreading on fibronectin and vitronectin. RPTPalpha-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPalpha is an early component in force-dependent signal transduction pathways leading to the assembly of focal complexes on both fibronectin and vitronectin.

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RPTPα deficiency does not affect integrin–ligand interactions, but leads to weak integrin–cytoskeleton linkages. (A) FN (left)- or VN (right)-coated beads (1-μm diam) bound specifically to the upper surface of RPTPα+/+ (+/+) and RPTPα−/− cells (−/−). Percentages of bound beads after 2 s were equalized with or without addition of GPen. Binding was compared with control beads coated with BSA. Results shown are the mean ± SD of three independent experiments. (B) Beads coated with FN (left) or VN (right) were placed on the upper surface of RPTPα+/+, RPTPα−/−, and RPTPα−/−wt cells, and the MSD was quantified after beads left the trap field (500 nm). Results shown are the mean ± SD of three independent experiments.
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fig5: RPTPα deficiency does not affect integrin–ligand interactions, but leads to weak integrin–cytoskeleton linkages. (A) FN (left)- or VN (right)-coated beads (1-μm diam) bound specifically to the upper surface of RPTPα+/+ (+/+) and RPTPα−/− cells (−/−). Percentages of bound beads after 2 s were equalized with or without addition of GPen. Binding was compared with control beads coated with BSA. Results shown are the mean ± SD of three independent experiments. (B) Beads coated with FN (left) or VN (right) were placed on the upper surface of RPTPα+/+, RPTPα−/−, and RPTPα−/−wt cells, and the MSD was quantified after beads left the trap field (500 nm). Results shown are the mean ± SD of three independent experiments.

Mentions: The sensitivity of cell spreading on ECM substrates to RPTPα expression raises the possibility that RPTPα also modulates integrin–ligand interactions (Hughes and Pfaff, 1998). To study more directly the role of RPTPα in regulating integrin–ligand interactions, we placed ligand-coated beads on the upper surface of spreading fibroblasts with a laser trap (<0.5 μm from the leading edge of the cell). This allows the quantification of changes in ligand binding and the dynamics of traction force generation by integrins (Choquet et al., 1997), which are parameters that are essential to cell spreading and migration (Lauffenburger and Horwitz, 1996). Beads were coated with a recombinant fragment of FN (FNIII7-10), purified human VN, or BSA. Beads coated with either FNIII7-10 or VN bound to the surface with a similar frequency and significantly better than BSA-coated control beads (Fig. 5 A). The binding of VN-coated beads was reduced to the level of control beads by pretreatment with GPen (Fig. 5 A). When FNIII7-10–coated beads were placed and held on the upper surface of cells in the presence of GPen, there was a significant reduction of bead binding on both RPTPα+/+ and RPTPα−/− cells (Fig. 5 A). Together, these results suggest that integrin–ligand interactions are not modulated by RPTPα activity. The contrast between the impaired cell spreading and the unaffected bead binding indicates that RPTPα may regulate the strength or dynamics of integrin–cytoskeleton interactions.


RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages.

von Wichert G, Jiang G, Kostic A, De Vos K, Sap J, Sheetz MP - J. Cell Biol. (2003)

RPTPα deficiency does not affect integrin–ligand interactions, but leads to weak integrin–cytoskeleton linkages. (A) FN (left)- or VN (right)-coated beads (1-μm diam) bound specifically to the upper surface of RPTPα+/+ (+/+) and RPTPα−/− cells (−/−). Percentages of bound beads after 2 s were equalized with or without addition of GPen. Binding was compared with control beads coated with BSA. Results shown are the mean ± SD of three independent experiments. (B) Beads coated with FN (left) or VN (right) were placed on the upper surface of RPTPα+/+, RPTPα−/−, and RPTPα−/−wt cells, and the MSD was quantified after beads left the trap field (500 nm). Results shown are the mean ± SD of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172891&req=5

fig5: RPTPα deficiency does not affect integrin–ligand interactions, but leads to weak integrin–cytoskeleton linkages. (A) FN (left)- or VN (right)-coated beads (1-μm diam) bound specifically to the upper surface of RPTPα+/+ (+/+) and RPTPα−/− cells (−/−). Percentages of bound beads after 2 s were equalized with or without addition of GPen. Binding was compared with control beads coated with BSA. Results shown are the mean ± SD of three independent experiments. (B) Beads coated with FN (left) or VN (right) were placed on the upper surface of RPTPα+/+, RPTPα−/−, and RPTPα−/−wt cells, and the MSD was quantified after beads left the trap field (500 nm). Results shown are the mean ± SD of three independent experiments.
Mentions: The sensitivity of cell spreading on ECM substrates to RPTPα expression raises the possibility that RPTPα also modulates integrin–ligand interactions (Hughes and Pfaff, 1998). To study more directly the role of RPTPα in regulating integrin–ligand interactions, we placed ligand-coated beads on the upper surface of spreading fibroblasts with a laser trap (<0.5 μm from the leading edge of the cell). This allows the quantification of changes in ligand binding and the dynamics of traction force generation by integrins (Choquet et al., 1997), which are parameters that are essential to cell spreading and migration (Lauffenburger and Horwitz, 1996). Beads were coated with a recombinant fragment of FN (FNIII7-10), purified human VN, or BSA. Beads coated with either FNIII7-10 or VN bound to the surface with a similar frequency and significantly better than BSA-coated control beads (Fig. 5 A). The binding of VN-coated beads was reduced to the level of control beads by pretreatment with GPen (Fig. 5 A). When FNIII7-10–coated beads were placed and held on the upper surface of cells in the presence of GPen, there was a significant reduction of bead binding on both RPTPα+/+ and RPTPα−/− cells (Fig. 5 A). Together, these results suggest that integrin–ligand interactions are not modulated by RPTPα activity. The contrast between the impaired cell spreading and the unaffected bead binding indicates that RPTPα may regulate the strength or dynamics of integrin–cytoskeleton interactions.

Bottom Line: We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha).RPTPalpha colocalizes with alphav-integrins at the leading edge during early spreading, and coimmunoprecipitates with alphav-integrins during spreading on fibronectin and vitronectin.RPTPalpha-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha). RPTPalpha colocalizes with alphav-integrins at the leading edge during early spreading, and coimmunoprecipitates with alphav-integrins during spreading on fibronectin and vitronectin. RPTPalpha-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPalpha is an early component in force-dependent signal transduction pathways leading to the assembly of focal complexes on both fibronectin and vitronectin.

Show MeSH
Related in: MedlinePlus