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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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Cell spreading depends on RPTPα and αv/β3-integrins. (A) Percentage of spread cells on either FN or VN was quantified 30 min after plating. (left) Spreading of RPTPα+/+ cells (+/+) compared with RPTPα−/− cells (−/−). (right) RPTPα−/−wt cells (−/−wt) compared with RPTPα−/−vec cells (−/−vec). Results shown are the mean ± SD of three to five independent experiments. (B) RPTPα+/+ and RPTPα−/− cells were plated, and the percentage of spread cells on LA was quantified 30 min after plating. Results shown are the mean ± SD of three independent experiments. (C) Equal amounts of protein from each cell line were analyzed by Western blotting using RPTPα, αv-, and β3-integrin antibodies. Micrographs shown are representative of at least three independent experiments. (D) RPTPα+/+ cells were plated with or without GPen (0.5 mM) and anti–αv- and anti–β3- integrin antibodies (25 μg/ml); the percentage of spread cells on VN was quantified after 30 min and compared with control substrates coated with BSA. Results shown are the mean ± SD of three independent experiments. (E) Percentage of spread cells in the presence of GPen or anti–αv- or anti–β3-integrin antibodies was compared after 30 min on FN. (left) Spreading of RPTPα+/+ cells compared with RPTPα−/− cells. (right) RPTPα−/−wt cells compared with RPTPα−/−vec cells. Results shown are the mean ± SD of three independent experiments.
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fig1: Cell spreading depends on RPTPα and αv/β3-integrins. (A) Percentage of spread cells on either FN or VN was quantified 30 min after plating. (left) Spreading of RPTPα+/+ cells (+/+) compared with RPTPα−/− cells (−/−). (right) RPTPα−/−wt cells (−/−wt) compared with RPTPα−/−vec cells (−/−vec). Results shown are the mean ± SD of three to five independent experiments. (B) RPTPα+/+ and RPTPα−/− cells were plated, and the percentage of spread cells on LA was quantified 30 min after plating. Results shown are the mean ± SD of three independent experiments. (C) Equal amounts of protein from each cell line were analyzed by Western blotting using RPTPα, αv-, and β3-integrin antibodies. Micrographs shown are representative of at least three independent experiments. (D) RPTPα+/+ cells were plated with or without GPen (0.5 mM) and anti–αv- and anti–β3- integrin antibodies (25 μg/ml); the percentage of spread cells on VN was quantified after 30 min and compared with control substrates coated with BSA. Results shown are the mean ± SD of three independent experiments. (E) Percentage of spread cells in the presence of GPen or anti–αv- or anti–β3-integrin antibodies was compared after 30 min on FN. (left) Spreading of RPTPα+/+ cells compared with RPTPα−/− cells. (right) RPTPα−/−wt cells compared with RPTPα−/−vec cells. Results shown are the mean ± SD of three independent experiments.

Mentions: To further characterize the role of RPTPα in integrin function, we compared the spreading of fibroblasts derived from RPTPα-expressing (RPTPα+/+) or RPTPα-deficient (RPTPα−/−) mice on FN and VN. Cell spreading on FN depended on the expression of RPTPα (Fig. 1 A; Su et al., 1999). On VN substrates, RPTPα+/+ cells or RPTPα−/− cells expressing wild-type RPTPα (RPTPα−/−wt) spread within 30 min after plating (Fig. 1 A). Although RPTPα−/−wt cells reached on average only a level of RPTPα protein expression of 55%, compared with RPTPα+/+ cells (as judged by scanning densitometry [unpublished data]; Fig. 1 C), this level restored the phenotype. In contrast, RPTPα−/− cells or empty vector (RPTPα−/−vec)-transfected cells plated on VN substrates or RPTPα+/+ cells plated on control substrates (BSA) showed significantly less spreading (Fig. 1, A and D). Interestingly, there was no difference in the spreading ability on laminin (LA), suggesting a substrate dependency (Fig. 1 B).


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)

Cell spreading depends on RPTPα and αv/β3-integrins. (A) Percentage of spread cells on either FN or VN was quantified 30 min after plating. (left) Spreading of RPTPα+/+ cells (+/+) compared with RPTPα−/− cells (−/−). (right) RPTPα−/−wt cells (−/−wt) compared with RPTPα−/−vec cells (−/−vec). Results shown are the mean ± SD of three to five independent experiments. (B) RPTPα+/+ and RPTPα−/− cells were plated, and the percentage of spread cells on LA was quantified 30 min after plating. Results shown are the mean ± SD of three independent experiments. (C) Equal amounts of protein from each cell line were analyzed by Western blotting using RPTPα, αv-, and β3-integrin antibodies. Micrographs shown are representative of at least three independent experiments. (D) RPTPα+/+ cells were plated with or without GPen (0.5 mM) and anti–αv- and anti–β3- integrin antibodies (25 μg/ml); the percentage of spread cells on VN was quantified after 30 min and compared with control substrates coated with BSA. Results shown are the mean ± SD of three independent experiments. (E) Percentage of spread cells in the presence of GPen or anti–αv- or anti–β3-integrin antibodies was compared after 30 min on FN. (left) Spreading of RPTPα+/+ cells compared with RPTPα−/− cells. (right) RPTPα−/−wt cells compared with RPTPα−/−vec cells. Results shown are the mean ± SD of three independent experiments.
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Related In: Results  -  Collection

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fig1: Cell spreading depends on RPTPα and αv/β3-integrins. (A) Percentage of spread cells on either FN or VN was quantified 30 min after plating. (left) Spreading of RPTPα+/+ cells (+/+) compared with RPTPα−/− cells (−/−). (right) RPTPα−/−wt cells (−/−wt) compared with RPTPα−/−vec cells (−/−vec). Results shown are the mean ± SD of three to five independent experiments. (B) RPTPα+/+ and RPTPα−/− cells were plated, and the percentage of spread cells on LA was quantified 30 min after plating. Results shown are the mean ± SD of three independent experiments. (C) Equal amounts of protein from each cell line were analyzed by Western blotting using RPTPα, αv-, and β3-integrin antibodies. Micrographs shown are representative of at least three independent experiments. (D) RPTPα+/+ cells were plated with or without GPen (0.5 mM) and anti–αv- and anti–β3- integrin antibodies (25 μg/ml); the percentage of spread cells on VN was quantified after 30 min and compared with control substrates coated with BSA. Results shown are the mean ± SD of three independent experiments. (E) Percentage of spread cells in the presence of GPen or anti–αv- or anti–β3-integrin antibodies was compared after 30 min on FN. (left) Spreading of RPTPα+/+ cells compared with RPTPα−/− cells. (right) RPTPα−/−wt cells compared with RPTPα−/−vec cells. Results shown are the mean ± SD of three independent experiments.
Mentions: To further characterize the role of RPTPα in integrin function, we compared the spreading of fibroblasts derived from RPTPα-expressing (RPTPα+/+) or RPTPα-deficient (RPTPα−/−) mice on FN and VN. Cell spreading on FN depended on the expression of RPTPα (Fig. 1 A; Su et al., 1999). On VN substrates, RPTPα+/+ cells or RPTPα−/− cells expressing wild-type RPTPα (RPTPα−/−wt) spread within 30 min after plating (Fig. 1 A). Although RPTPα−/−wt cells reached on average only a level of RPTPα protein expression of 55%, compared with RPTPα+/+ cells (as judged by scanning densitometry [unpublished data]; Fig. 1 C), this level restored the phenotype. In contrast, RPTPα−/− cells or empty vector (RPTPα−/−vec)-transfected cells plated on VN substrates or RPTPα+/+ cells plated on control substrates (BSA) showed significantly less spreading (Fig. 1, A and D). Interestingly, there was no difference in the spreading ability on laminin (LA), suggesting a substrate dependency (Fig. 1 B).

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