Limits...
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.

Show MeSH

Related in: MedlinePlus

Response to force requires expression of RPTPα. (A, top) Accumulation of GFP-paxillin in RPTPα+/+ cells (+/+) in serial micrographs of rearward moving beads coated with FN (1-μm diam) after placement on the upper surface and escape out of the trap. Beads position is indicated by an arrow. (bottom, left) Beads were placed on the upper surface and GFP-paxillin assembly was quantified without application of force in RPTPα+/+ cells. (bottom, right) Serial micrographs of RPTPα+/+ cells transfected with EGFP alone (left) and of GFP-paxillin transfected RPTPα+/+ cells after placement of Con A–coated beads (right). (B) Time-lapse micrographs of GFP-paxillin–expressing RPTPα−/− cells (−/−) after placement and escape out of the trap of FN-coated beads. (C) Time-lapse micrographs of rearward moving FN beads after placement and escape out of the trap on RPTPα−/−wt cells (−/−wt). (D) Model for the force-dependent assembly of focal complexes. First, upon formation of active lamellipodia, a complex of αv/β3-integrins and RPTPα is formed, localizing to the edge of the lamellipodium. Second, force application to αv/β3-integrins leads to RPTPα-dependent activation of SFK. Third, SFK-activation promotes the assembly and the reinforcement of focal complexes at early times. Finally, as focal complexes mature, SFK activity is also required for turnover of adhesion sites.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172891&req=5

fig7: Response to force requires expression of RPTPα. (A, top) Accumulation of GFP-paxillin in RPTPα+/+ cells (+/+) in serial micrographs of rearward moving beads coated with FN (1-μm diam) after placement on the upper surface and escape out of the trap. Beads position is indicated by an arrow. (bottom, left) Beads were placed on the upper surface and GFP-paxillin assembly was quantified without application of force in RPTPα+/+ cells. (bottom, right) Serial micrographs of RPTPα+/+ cells transfected with EGFP alone (left) and of GFP-paxillin transfected RPTPα+/+ cells after placement of Con A–coated beads (right). (B) Time-lapse micrographs of GFP-paxillin–expressing RPTPα−/− cells (−/−) after placement and escape out of the trap of FN-coated beads. (C) Time-lapse micrographs of rearward moving FN beads after placement and escape out of the trap on RPTPα−/−wt cells (−/−wt). (D) Model for the force-dependent assembly of focal complexes. First, upon formation of active lamellipodia, a complex of αv/β3-integrins and RPTPα is formed, localizing to the edge of the lamellipodium. Second, force application to αv/β3-integrins leads to RPTPα-dependent activation of SFK. Third, SFK-activation promotes the assembly and the reinforcement of focal complexes at early times. Finally, as focal complexes mature, SFK activity is also required for turnover of adhesion sites.

Mentions: It has been suggested that phosphatases are involved in the process of mechanotransduction and our data are consistent with the idea that the phosphatase RPTPα is required for force-dependent reinforcement of integrin–cytoskeleton linkages. To further address this hypothesis, we designed a laser trap experiment that allowed the visualization of GFP-tagged protein dynamics in living cells and tested whether the application of force to small ligand-coated beads (1-μm diam) induces the accumulation of focal contact proteins to the adhesion site. As predicted, there was no accumulation of paxillin to the binding site in RPTPα+/+ cells when the trap was turned off after placement of beads (0%, n = 25; Fig. 7 A, bottom left). Strikingly, the restraint of FNIII7-10-coated beads with the trap caused accumulation of GFP-paxillin to the binding site within seconds in RPTPα+/+ cells (82%, n = 22; Fig. 7 A, top). The paxillin accumulation began as a distinct spot underneath the bead after application of force. However, as the bead was pulled out of the trap by the retrograde motion of the cytoskeleton, force was exerted on the bead, and the pattern of paxillin assembly changed to a ring around the bead (as seen with the large beads). When RPTPα−/− cells were tested, significantly fewer cells accumulated paxillin to the site of interaction, with (15%, n = 20) or without (0%, n = 24; unpublished data) sustained application of force by the trap (Fig. 7 B). To confirm a dependency on the expression of RPTPα, we performed these experiments with RPTPα−/−wt cells. As expected, reexpression of RPTPα restored the ability to respond to applied forces with the accumulation of paxillin (65%, n = 23; Fig. 7 C). These data strongly indicate that RPTPα is part of force-dependent signal transduction events, and that it is a crucial component in this process.


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)

Response to force requires expression of RPTPα. (A, top) Accumulation of GFP-paxillin in RPTPα+/+ cells (+/+) in serial micrographs of rearward moving beads coated with FN (1-μm diam) after placement on the upper surface and escape out of the trap. Beads position is indicated by an arrow. (bottom, left) Beads were placed on the upper surface and GFP-paxillin assembly was quantified without application of force in RPTPα+/+ cells. (bottom, right) Serial micrographs of RPTPα+/+ cells transfected with EGFP alone (left) and of GFP-paxillin transfected RPTPα+/+ cells after placement of Con A–coated beads (right). (B) Time-lapse micrographs of GFP-paxillin–expressing RPTPα−/− cells (−/−) after placement and escape out of the trap of FN-coated beads. (C) Time-lapse micrographs of rearward moving FN beads after placement and escape out of the trap on RPTPα−/−wt cells (−/−wt). (D) Model for the force-dependent assembly of focal complexes. First, upon formation of active lamellipodia, a complex of αv/β3-integrins and RPTPα is formed, localizing to the edge of the lamellipodium. Second, force application to αv/β3-integrins leads to RPTPα-dependent activation of SFK. Third, SFK-activation promotes the assembly and the reinforcement of focal complexes at early times. Finally, as focal complexes mature, SFK activity is also required for turnover of adhesion sites.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Response to force requires expression of RPTPα. (A, top) Accumulation of GFP-paxillin in RPTPα+/+ cells (+/+) in serial micrographs of rearward moving beads coated with FN (1-μm diam) after placement on the upper surface and escape out of the trap. Beads position is indicated by an arrow. (bottom, left) Beads were placed on the upper surface and GFP-paxillin assembly was quantified without application of force in RPTPα+/+ cells. (bottom, right) Serial micrographs of RPTPα+/+ cells transfected with EGFP alone (left) and of GFP-paxillin transfected RPTPα+/+ cells after placement of Con A–coated beads (right). (B) Time-lapse micrographs of GFP-paxillin–expressing RPTPα−/− cells (−/−) after placement and escape out of the trap of FN-coated beads. (C) Time-lapse micrographs of rearward moving FN beads after placement and escape out of the trap on RPTPα−/−wt cells (−/−wt). (D) Model for the force-dependent assembly of focal complexes. First, upon formation of active lamellipodia, a complex of αv/β3-integrins and RPTPα is formed, localizing to the edge of the lamellipodium. Second, force application to αv/β3-integrins leads to RPTPα-dependent activation of SFK. Third, SFK-activation promotes the assembly and the reinforcement of focal complexes at early times. Finally, as focal complexes mature, SFK activity is also required for turnover of adhesion sites.
Mentions: It has been suggested that phosphatases are involved in the process of mechanotransduction and our data are consistent with the idea that the phosphatase RPTPα is required for force-dependent reinforcement of integrin–cytoskeleton linkages. To further address this hypothesis, we designed a laser trap experiment that allowed the visualization of GFP-tagged protein dynamics in living cells and tested whether the application of force to small ligand-coated beads (1-μm diam) induces the accumulation of focal contact proteins to the adhesion site. As predicted, there was no accumulation of paxillin to the binding site in RPTPα+/+ cells when the trap was turned off after placement of beads (0%, n = 25; Fig. 7 A, bottom left). Strikingly, the restraint of FNIII7-10-coated beads with the trap caused accumulation of GFP-paxillin to the binding site within seconds in RPTPα+/+ cells (82%, n = 22; Fig. 7 A, top). The paxillin accumulation began as a distinct spot underneath the bead after application of force. However, as the bead was pulled out of the trap by the retrograde motion of the cytoskeleton, force was exerted on the bead, and the pattern of paxillin assembly changed to a ring around the bead (as seen with the large beads). When RPTPα−/− cells were tested, significantly fewer cells accumulated paxillin to the site of interaction, with (15%, n = 20) or without (0%, n = 24; unpublished data) sustained application of force by the trap (Fig. 7 B). To confirm a dependency on the expression of RPTPα, we performed these experiments with RPTPα−/−wt cells. As expected, reexpression of RPTPα restored the ability to respond to applied forces with the accumulation of paxillin (65%, n = 23; Fig. 7 C). These data strongly indicate that RPTPα is part of force-dependent signal transduction events, and that it is a crucial component in this process.

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