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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 and αv/β3-integrins form a complex and cooperate in the activation of SFK. (A) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated for 15 min on FN or VN with or without GPen. Alternatively, cells were plated for 240 min on FN or were kept in suspension for 60 min. Cells were subsequently cross-linked, lysed, and subjected to immunoprecipitation with (+) or without (−) addition of anti–αv- or anti–α5-integrin antibodies. Western blotting was performed with either anti-RPTPα (top) or anti–αv- and α5- integrin (bottom) antibodies. Micrographs shown are representatives of at least two independent experiments. (B, top) RPTPα+/+ (+/+), RPTPα−/− (−/−), and RPTPα−/−wt cells (−/−wt) were spread for 15 min on FN or VN with or without GPen, lysed, and equal amounts of protein were analyzed by Western blotting using a phosphospecific anti-SFK antibody (Tyr416), a de-phosphospecific anti–c-Src antibody (Tyr 527) or an anti–c-Src antibody. (bottom) Relative Tyr416 autophosphorylation of SFK was quantified using scanning densitometry and normalized to control cells (+/+). Results shown are the mean ± SD of three independent experiments.
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fig3: RPTPα and αv/β3-integrins form a complex and cooperate in the activation of SFK. (A) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated for 15 min on FN or VN with or without GPen. Alternatively, cells were plated for 240 min on FN or were kept in suspension for 60 min. Cells were subsequently cross-linked, lysed, and subjected to immunoprecipitation with (+) or without (−) addition of anti–αv- or anti–α5-integrin antibodies. Western blotting was performed with either anti-RPTPα (top) or anti–αv- and α5- integrin (bottom) antibodies. Micrographs shown are representatives of at least two independent experiments. (B, top) RPTPα+/+ (+/+), RPTPα−/− (−/−), and RPTPα−/−wt cells (−/−wt) were spread for 15 min on FN or VN with or without GPen, lysed, and equal amounts of protein were analyzed by Western blotting using a phosphospecific anti-SFK antibody (Tyr416), a de-phosphospecific anti–c-Src antibody (Tyr 527) or an anti–c-Src antibody. (bottom) Relative Tyr416 autophosphorylation of SFK was quantified using scanning densitometry and normalized to control cells (+/+). Results shown are the mean ± SD of three independent experiments.

Mentions: To further investigate the relationship between RPTPα and αv/β3-integrins, we performed coimmunoprecipitation studies. We were able to coimmunoprecipitate RPTPα in anti–αv-integrin immunoprecipitates of spreading RPTPα+/+ cells after pretreatment with a water-soluble cross-linker on both FN and VN. This association was restricted to the early phases of spreading because there was no coimmunoprecipitation after 240 min. Furthermore, activation of αv/β3-integrins was necessary for the formation of the complex because cells kept in suspension for 60 min or cells pretreated with GPen did not show association of RPTPα with αv/β3-integrins. In addition, this interaction was specific for αv-integrins because anti–α5-integrin immunoprecipitates did not contain RPTPα (Fig. 3 A).


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 and αv/β3-integrins form a complex and cooperate in the activation of SFK. (A) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated for 15 min on FN or VN with or without GPen. Alternatively, cells were plated for 240 min on FN or were kept in suspension for 60 min. Cells were subsequently cross-linked, lysed, and subjected to immunoprecipitation with (+) or without (−) addition of anti–αv- or anti–α5-integrin antibodies. Western blotting was performed with either anti-RPTPα (top) or anti–αv- and α5- integrin (bottom) antibodies. Micrographs shown are representatives of at least two independent experiments. (B, top) RPTPα+/+ (+/+), RPTPα−/− (−/−), and RPTPα−/−wt cells (−/−wt) were spread for 15 min on FN or VN with or without GPen, lysed, and equal amounts of protein were analyzed by Western blotting using a phosphospecific anti-SFK antibody (Tyr416), a de-phosphospecific anti–c-Src antibody (Tyr 527) or an anti–c-Src antibody. (bottom) Relative Tyr416 autophosphorylation of SFK was quantified using scanning densitometry and normalized to control cells (+/+). Results shown are the mean ± SD of three independent experiments.
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Related In: Results  -  Collection

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fig3: RPTPα and αv/β3-integrins form a complex and cooperate in the activation of SFK. (A) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated for 15 min on FN or VN with or without GPen. Alternatively, cells were plated for 240 min on FN or were kept in suspension for 60 min. Cells were subsequently cross-linked, lysed, and subjected to immunoprecipitation with (+) or without (−) addition of anti–αv- or anti–α5-integrin antibodies. Western blotting was performed with either anti-RPTPα (top) or anti–αv- and α5- integrin (bottom) antibodies. Micrographs shown are representatives of at least two independent experiments. (B, top) RPTPα+/+ (+/+), RPTPα−/− (−/−), and RPTPα−/−wt cells (−/−wt) were spread for 15 min on FN or VN with or without GPen, lysed, and equal amounts of protein were analyzed by Western blotting using a phosphospecific anti-SFK antibody (Tyr416), a de-phosphospecific anti–c-Src antibody (Tyr 527) or an anti–c-Src antibody. (bottom) Relative Tyr416 autophosphorylation of SFK was quantified using scanning densitometry and normalized to control cells (+/+). Results shown are the mean ± SD of three independent experiments.
Mentions: To further investigate the relationship between RPTPα and αv/β3-integrins, we performed coimmunoprecipitation studies. We were able to coimmunoprecipitate RPTPα in anti–αv-integrin immunoprecipitates of spreading RPTPα+/+ cells after pretreatment with a water-soluble cross-linker on both FN and VN. This association was restricted to the early phases of spreading because there was no coimmunoprecipitation after 240 min. Furthermore, activation of αv/β3-integrins was necessary for the formation of the complex because cells kept in suspension for 60 min or cells pretreated with GPen did not show association of RPTPα with αv/β3-integrins. In addition, this interaction was specific for αv-integrins because anti–α5-integrin immunoprecipitates did not contain RPTPα (Fig. 3 A).

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