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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 colocalizes with αv/β3-integrins at early times. (A) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 and 240 min, fixed and stained with anti–αv-integrin and paxillin antibodies. Distribution was analyzed on either FN (a–j) or VN (k–t) after 30 min (left column) and 240 min (right column). Merges show the overlay of RPTPα and αv-integrins (c, h, m, and r) and the overlay of RPTPα, αv-integrins, and paxillin (e, j, o, and t). Micrographs shown are representative of at least five independent experiments. (B) RPTPα+/+ cells (+/+) were transiently transfected with EGFP, and RPTPα−/− cells (−/−) were transiently transfected with either EGFP or RPTPα-YFP and spread for 30 min, and the percentage of spread fluorescent cells on either FN (left) or VN (right) was quantified. Results shown are the mean ± SD of three independent experiments. (C) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 min on LA. Picture shown is representative of two independent experiments.
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fig2: RPTPα colocalizes with αv/β3-integrins at early times. (A) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 and 240 min, fixed and stained with anti–αv-integrin and paxillin antibodies. Distribution was analyzed on either FN (a–j) or VN (k–t) after 30 min (left column) and 240 min (right column). Merges show the overlay of RPTPα and αv-integrins (c, h, m, and r) and the overlay of RPTPα, αv-integrins, and paxillin (e, j, o, and t). Micrographs shown are representative of at least five independent experiments. (B) RPTPα+/+ cells (+/+) were transiently transfected with EGFP, and RPTPα−/− cells (−/−) were transiently transfected with either EGFP or RPTPα-YFP and spread for 30 min, and the percentage of spread fluorescent cells on either FN (left) or VN (right) was quantified. Results shown are the mean ± SD of three independent experiments. (C) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 min on LA. Picture shown is representative of two independent experiments.

Mentions: Next, we wanted to determine whether RPTPα colocalizes with αv/β3-integrins. RPTPα−/− cells were transiently transfected with an RPTPα construct in which the D2 phosphatase domain was replaced by YFP (Buist et al., 2000). To confirm functionality, we performed spreading assays with RPTPα-YFP– transfected RPTPα−/− cells and found a spreading ability similar to the RPTPα+/+ cells (Fig. 2 B). Indirect immunofluorescence was used to visualize the αv-integrins and paxillin. Confocal microscopy revealed that RPTPα colocalized with αv-integrins and paxillin at the leading edge on both FN and VN 30 min after plating in spreading cells (Fig. 2 A, a–e and k–o). After 240 min, all cells were spread and RPTPα was diffusely localized in the membrane (Fig. 2 A, f–j and p–t), whereas colocalization of αv-integrins with paxillin occurred in the area of focal contacts (Fig. 2 A, j and t). On LA, there was no localization of RPTPα to the leading edge in spreading cells (Fig. 2 C). Thus, we find evidence of a transient association between RPTPα and αv/β3-integrins during the early phases of focal complex formation on both FN and VN.


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 colocalizes with αv/β3-integrins at early times. (A) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 and 240 min, fixed and stained with anti–αv-integrin and paxillin antibodies. Distribution was analyzed on either FN (a–j) or VN (k–t) after 30 min (left column) and 240 min (right column). Merges show the overlay of RPTPα and αv-integrins (c, h, m, and r) and the overlay of RPTPα, αv-integrins, and paxillin (e, j, o, and t). Micrographs shown are representative of at least five independent experiments. (B) RPTPα+/+ cells (+/+) were transiently transfected with EGFP, and RPTPα−/− cells (−/−) were transiently transfected with either EGFP or RPTPα-YFP and spread for 30 min, and the percentage of spread fluorescent cells on either FN (left) or VN (right) was quantified. Results shown are the mean ± SD of three independent experiments. (C) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 min on LA. Picture shown is representative of two independent experiments.
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fig2: RPTPα colocalizes with αv/β3-integrins at early times. (A) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 and 240 min, fixed and stained with anti–αv-integrin and paxillin antibodies. Distribution was analyzed on either FN (a–j) or VN (k–t) after 30 min (left column) and 240 min (right column). Merges show the overlay of RPTPα and αv-integrins (c, h, m, and r) and the overlay of RPTPα, αv-integrins, and paxillin (e, j, o, and t). Micrographs shown are representative of at least five independent experiments. (B) RPTPα+/+ cells (+/+) were transiently transfected with EGFP, and RPTPα−/− cells (−/−) were transiently transfected with either EGFP or RPTPα-YFP and spread for 30 min, and the percentage of spread fluorescent cells on either FN (left) or VN (right) was quantified. Results shown are the mean ± SD of three independent experiments. (C) RPTPα−/− cells were transiently transfected with RPTPα-YFP and spread for 30 min on LA. Picture shown is representative of two independent experiments.
Mentions: Next, we wanted to determine whether RPTPα colocalizes with αv/β3-integrins. RPTPα−/− cells were transiently transfected with an RPTPα construct in which the D2 phosphatase domain was replaced by YFP (Buist et al., 2000). To confirm functionality, we performed spreading assays with RPTPα-YFP– transfected RPTPα−/− cells and found a spreading ability similar to the RPTPα+/+ cells (Fig. 2 B). Indirect immunofluorescence was used to visualize the αv-integrins and paxillin. Confocal microscopy revealed that RPTPα colocalized with αv-integrins and paxillin at the leading edge on both FN and VN 30 min after plating in spreading cells (Fig. 2 A, a–e and k–o). After 240 min, all cells were spread and RPTPα was diffusely localized in the membrane (Fig. 2 A, f–j and p–t), whereas colocalization of αv-integrins with paxillin occurred in the area of focal contacts (Fig. 2 A, j and t). On LA, there was no localization of RPTPα to the leading edge in spreading cells (Fig. 2 C). Thus, we find evidence of a transient association between RPTPα and αv/β3-integrins during the early phases of focal complex formation on both FN and VN.

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