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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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Formation of focal complexes through RPTPα and αv/β3-integrins via SFK. (A, top) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transfected with GFP-paxillin and spread for 30 min on FN or VN. The distribution of GFP-paxillin was compared 30 min after plating (a, d, g, and j). Cells were pretreated with GPen (b, e, h, and k) or cotransfected with CSK (c, f, i, and l), and the number of cells with either focal complexes or contacts was quantified. (bottom, left) RPTPα−/−wt cells (−/−wt) were allowed to spread on FN or VN and paxillin assembly in distinct adhesion sites was quantified after 30 min. (bottom, right) RPTPα+/+ cells were cotransfected with (+CSK) or without CSK and GFP-paxillin, fixed, and stained with anti–phospho-SFK. Micrographs shown are representative of at least three independent experiments. (B) Percentage of spread cells showing GFP-paxillin assembly in distinct adhesion sites was quantified versus cells exhibiting cytoplasmic distribution of GFP-paxillin on either FN (top) or VN (bottom). Results shown are the mean ± SD of five independent experiments. (C) Wild-type cells (c-Src+/+) (+/+) or c-Src-Fyn-Yes–deficient cells (SYF) were plated on FN or VN for 15 min, and paxillin assembly in distinct adhesion sites was quantified. Results shown are the mean ± SD of three independent experiments. (D) SYF cells were plated on FN for 15 min with or without cotransfection of GFP-paxillin with wild-type Fyn, c-Src, or c-Yes. GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of three independent experiments. (E, left) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated on FN for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. The percentage of cells showing GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of at least five independent experiments. (right) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transiently transfected with wild-type Fyn, c-Src, or c-Yes, subsequently lysed, and equal amounts of protein were analyzed by Western blotting using anti-Fyn, c-Src, or c-Yes antibodies or a phosphospecific anti-SFK antibody (Tyr416). Membranes were stripped and probed for actin to confirm protein content. Micrographs shown are representative of two independent experiments.
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fig4: Formation of focal complexes through RPTPα and αv/β3-integrins via SFK. (A, top) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transfected with GFP-paxillin and spread for 30 min on FN or VN. The distribution of GFP-paxillin was compared 30 min after plating (a, d, g, and j). Cells were pretreated with GPen (b, e, h, and k) or cotransfected with CSK (c, f, i, and l), and the number of cells with either focal complexes or contacts was quantified. (bottom, left) RPTPα−/−wt cells (−/−wt) were allowed to spread on FN or VN and paxillin assembly in distinct adhesion sites was quantified after 30 min. (bottom, right) RPTPα+/+ cells were cotransfected with (+CSK) or without CSK and GFP-paxillin, fixed, and stained with anti–phospho-SFK. Micrographs shown are representative of at least three independent experiments. (B) Percentage of spread cells showing GFP-paxillin assembly in distinct adhesion sites was quantified versus cells exhibiting cytoplasmic distribution of GFP-paxillin on either FN (top) or VN (bottom). Results shown are the mean ± SD of five independent experiments. (C) Wild-type cells (c-Src+/+) (+/+) or c-Src-Fyn-Yes–deficient cells (SYF) were plated on FN or VN for 15 min, and paxillin assembly in distinct adhesion sites was quantified. Results shown are the mean ± SD of three independent experiments. (D) SYF cells were plated on FN for 15 min with or without cotransfection of GFP-paxillin with wild-type Fyn, c-Src, or c-Yes. GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of three independent experiments. (E, left) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated on FN for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. The percentage of cells showing GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of at least five independent experiments. (right) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transiently transfected with wild-type Fyn, c-Src, or c-Yes, subsequently lysed, and equal amounts of protein were analyzed by Western blotting using anti-Fyn, c-Src, or c-Yes antibodies or a phosphospecific anti-SFK antibody (Tyr416). Membranes were stripped and probed for actin to confirm protein content. Micrographs shown are representative of two independent experiments.

Mentions: To study more directly the interplay between RPTPα and αv/β3-integrins, we observed the distribution of GFP-tagged paxillin on different substrates. Paxillin is a focal adhesion–associated adaptor protein that is a known marker for focal adhesions and focal complexes (Beningo et al., 2001). GFP-paxillin distribution in distinct adhesion sites versus cytoplasmic localization was analyzed in spread cells 30 min after plating. In RPTPα+/+ cells on both substrates, GFP-paxillin distributed to peripheral stripes in the majority of cells (Fig. 4, A [a and g] and B). In contrast, in RPTPα−/− cells, there was more than twofold lower focal complex or contact formation at early times. GFP-paxillin remained mainly cytoplasmic and localized to the leading edge (Fig. 4, A [d and j] and B). To determine the function of the αv/β3-integrin in this process, we plated GFP-paxillin–transfected cells on FN and VN in the presence of GPen. In RPTPα+/+ cells on both substrates, there was significantly less formation of distinct adhesion sites with GPen, as judged by paxillin assembly (Fig. 4, A [b and h] and B). In contrast, there was no further reduction in the number of cells exhibiting formation of adhesion sites in the RPTPα−/− cells with GPen (Fig. 4, A [e and k] and B). As expected, GPen drastically reduced the ability of both RPTPα+/+ and RPTPα−/− cells to spread on VN (Fig. 4, A [h and k] and 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)

Formation of focal complexes through RPTPα and αv/β3-integrins via SFK. (A, top) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transfected with GFP-paxillin and spread for 30 min on FN or VN. The distribution of GFP-paxillin was compared 30 min after plating (a, d, g, and j). Cells were pretreated with GPen (b, e, h, and k) or cotransfected with CSK (c, f, i, and l), and the number of cells with either focal complexes or contacts was quantified. (bottom, left) RPTPα−/−wt cells (−/−wt) were allowed to spread on FN or VN and paxillin assembly in distinct adhesion sites was quantified after 30 min. (bottom, right) RPTPα+/+ cells were cotransfected with (+CSK) or without CSK and GFP-paxillin, fixed, and stained with anti–phospho-SFK. Micrographs shown are representative of at least three independent experiments. (B) Percentage of spread cells showing GFP-paxillin assembly in distinct adhesion sites was quantified versus cells exhibiting cytoplasmic distribution of GFP-paxillin on either FN (top) or VN (bottom). Results shown are the mean ± SD of five independent experiments. (C) Wild-type cells (c-Src+/+) (+/+) or c-Src-Fyn-Yes–deficient cells (SYF) were plated on FN or VN for 15 min, and paxillin assembly in distinct adhesion sites was quantified. Results shown are the mean ± SD of three independent experiments. (D) SYF cells were plated on FN for 15 min with or without cotransfection of GFP-paxillin with wild-type Fyn, c-Src, or c-Yes. GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of three independent experiments. (E, left) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated on FN for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. The percentage of cells showing GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of at least five independent experiments. (right) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transiently transfected with wild-type Fyn, c-Src, or c-Yes, subsequently lysed, and equal amounts of protein were analyzed by Western blotting using anti-Fyn, c-Src, or c-Yes antibodies or a phosphospecific anti-SFK antibody (Tyr416). Membranes were stripped and probed for actin to confirm protein content. Micrographs shown are representative of two independent experiments.
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fig4: Formation of focal complexes through RPTPα and αv/β3-integrins via SFK. (A, top) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transfected with GFP-paxillin and spread for 30 min on FN or VN. The distribution of GFP-paxillin was compared 30 min after plating (a, d, g, and j). Cells were pretreated with GPen (b, e, h, and k) or cotransfected with CSK (c, f, i, and l), and the number of cells with either focal complexes or contacts was quantified. (bottom, left) RPTPα−/−wt cells (−/−wt) were allowed to spread on FN or VN and paxillin assembly in distinct adhesion sites was quantified after 30 min. (bottom, right) RPTPα+/+ cells were cotransfected with (+CSK) or without CSK and GFP-paxillin, fixed, and stained with anti–phospho-SFK. Micrographs shown are representative of at least three independent experiments. (B) Percentage of spread cells showing GFP-paxillin assembly in distinct adhesion sites was quantified versus cells exhibiting cytoplasmic distribution of GFP-paxillin on either FN (top) or VN (bottom). Results shown are the mean ± SD of five independent experiments. (C) Wild-type cells (c-Src+/+) (+/+) or c-Src-Fyn-Yes–deficient cells (SYF) were plated on FN or VN for 15 min, and paxillin assembly in distinct adhesion sites was quantified. Results shown are the mean ± SD of three independent experiments. (D) SYF cells were plated on FN for 15 min with or without cotransfection of GFP-paxillin with wild-type Fyn, c-Src, or c-Yes. GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of three independent experiments. (E, left) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were plated on FN for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. The percentage of cells showing GFP-paxillin assembly in distinct adhesion sites was quantified as described above. Results shown are the mean ± SD of at least five independent experiments. (right) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were transiently transfected with wild-type Fyn, c-Src, or c-Yes, subsequently lysed, and equal amounts of protein were analyzed by Western blotting using anti-Fyn, c-Src, or c-Yes antibodies or a phosphospecific anti-SFK antibody (Tyr416). Membranes were stripped and probed for actin to confirm protein content. Micrographs shown are representative of two independent experiments.
Mentions: To study more directly the interplay between RPTPα and αv/β3-integrins, we observed the distribution of GFP-tagged paxillin on different substrates. Paxillin is a focal adhesion–associated adaptor protein that is a known marker for focal adhesions and focal complexes (Beningo et al., 2001). GFP-paxillin distribution in distinct adhesion sites versus cytoplasmic localization was analyzed in spread cells 30 min after plating. In RPTPα+/+ cells on both substrates, GFP-paxillin distributed to peripheral stripes in the majority of cells (Fig. 4, A [a and g] and B). In contrast, in RPTPα−/− cells, there was more than twofold lower focal complex or contact formation at early times. GFP-paxillin remained mainly cytoplasmic and localized to the leading edge (Fig. 4, A [d and j] and B). To determine the function of the αv/β3-integrin in this process, we plated GFP-paxillin–transfected cells on FN and VN in the presence of GPen. In RPTPα+/+ cells on both substrates, there was significantly less formation of distinct adhesion sites with GPen, as judged by paxillin assembly (Fig. 4, A [b and h] and B). In contrast, there was no further reduction in the number of cells exhibiting formation of adhesion sites in the RPTPα−/− cells with GPen (Fig. 4, A [e and k] and B). As expected, GPen drastically reduced the ability of both RPTPα+/+ and RPTPα−/− cells to spread on VN (Fig. 4, A [h and k] and 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