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

Reinforcement requires RPTPα- and αv/β3-integrin–dependent activation of SFK. (A, top) RPTPα+/+ and RPTPα−/− cells were transfected with GFP-paxillin and were spread for 30 min. Large beads (5.9-μm diam) coated with FN were spun onto the cells and incubated for 30 min. The fraction of bound beads causing accumulation (fluorescence intensity >2× surrounding; inset) of paxillin was determined. Confocal stacks were resliced along the indicated line over the beads position (bottom of each panel), shown are overlays of GFP-paxillin fluorescence intensity in pseudo-colors and the differential interference contrast image. (middle) RPTPα+/+ and RPTPα−/− cells were pretreated with GPen. (bottom) RPTPα+/+ and RPTPα−/− cells were cotransfected with CSK and analyzed as described above. (B, top) Percentage of FN- or Con A–coated beads causing accumulation of paxillin. Results shown are the mean ± SD of three independent experiments. (bottom) RPTPα−/−wt cells (−/−wt) 30 min after application of beads. (bottom, right) RPTPα+/+ cells transfected with EGFP alone 30 min after application of beads. (C) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were spread for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. Accumulation was analyzed as described above. (left) Percentage of FN-coated beads causing accumulation of paxillin. (right) Representative micrographs of RPTPα−/− cells coexpressing Fyn (top), c-Src (bottom, left), c-Yes (bottom, right), and GFP-paxillin 30 min after application of FN-coated beads.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172891&req=5

fig6: Reinforcement requires RPTPα- and αv/β3-integrin–dependent activation of SFK. (A, top) RPTPα+/+ and RPTPα−/− cells were transfected with GFP-paxillin and were spread for 30 min. Large beads (5.9-μm diam) coated with FN were spun onto the cells and incubated for 30 min. The fraction of bound beads causing accumulation (fluorescence intensity >2× surrounding; inset) of paxillin was determined. Confocal stacks were resliced along the indicated line over the beads position (bottom of each panel), shown are overlays of GFP-paxillin fluorescence intensity in pseudo-colors and the differential interference contrast image. (middle) RPTPα+/+ and RPTPα−/− cells were pretreated with GPen. (bottom) RPTPα+/+ and RPTPα−/− cells were cotransfected with CSK and analyzed as described above. (B, top) Percentage of FN- or Con A–coated beads causing accumulation of paxillin. Results shown are the mean ± SD of three independent experiments. (bottom) RPTPα−/−wt cells (−/−wt) 30 min after application of beads. (bottom, right) RPTPα+/+ cells transfected with EGFP alone 30 min after application of beads. (C) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were spread for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. Accumulation was analyzed as described above. (left) Percentage of FN-coated beads causing accumulation of paxillin. (right) Representative micrographs of RPTPα−/− cells coexpressing Fyn (top), c-Src (bottom, left), c-Yes (bottom, right), and GFP-paxillin 30 min after application of FN-coated beads.

Mentions: To further analyze the reinforcement process, we centrifuged onto cells (5 min/50 g) large beads (5.9-μm diam) coated with either FNIII7-10 or human VN. In contrast to the small beads used for the laser trap experiments, the large beads can cause the establishment of adhesion sites and, therefore, reinforcement by cellular contractions independent of laser trap restraint (Galbraith et al., 2002). Interestingly, 74% of the FNIII7-10–coated beads showed accumulation of GFP-paxillin to the bead periphery after 30 min on RPTPα+/+ cells (46% of VN-coated beads; unpublished data). In contrast, only 14% of the FNIII7-10–coated beads (7% of VN-coated beads; unpublished data) on RPTPα−/− cells showed redistribution of paxillin to the binding site (Fig. 6, A and B) . Next, we wanted to examine the function of SFK and αv/β3-integrins in the reinforcement process. Interestingly, pretreatment with GPen reduced the fraction of FNIII7-10–coated beads accumulating paxillin on RPTPα+/+ cells to 18%, but caused no significant reduction in the number of accumulating beads on the RPTPα−/− cells (11%; Fig. 6, A and B). Because the pretreatment with GPen prevented specific binding of VN-coated beads to the upper surface of RPTPα+/+ and RPTPα−/− cells (Fig. 5 A), there was no accumulation of paxillin around VN-coated beads in any case (unpublished data). Coexpression of CSK with paxillin decreased the number of FNIII7-10–coated beads, causing accumulation of paxillin to 16% in RPTPα+/+ cells, whereas there was no further reduction in RPTPα−/− cells (8%; Fig. 6, A and B). Consistent results could be obtained with VN-coated beads (8% for RPTPα+/+ cells and 5% for RPTPα−/− cells; unpublished data). We have shown earlier that RPTPα-dependent activation of Fyn is needed for the formation of focal complexes and contacts, suggesting that it might affect reinforcement. Indeed, coexpression of Fyn with GFP-paxillin led to a significant increase in the number of RPTPα−/− cells accumulating paxillin around beads (68%). In clear contrast, the coexpression of c-Src or c-Yes did not increase the number of accumulating beads in RPTPα−/− cells, but coexpression of c-Src reduced the number of cells responding to the beads in RPTPα+/+ cells to 34% (Fig. 6 C). Reintroduction of wild-type RPTPα into RPTPα−/− cells restored the ability to assemble paxillin at the binding site (Fig. 6 B). To confirm ligand specificity, we used Con A–coated beads on RPTPα+/+ cells, which showed accumulation of GFP-paxillin at the site of adhesion in only 7% of the beads, possibly due to unspecific activation of integrin receptors (Fig. 6 B). To exclude the possibility of a volume effect around the beads, we transfected RPTPα+/+ cells with EGFP alone, which did not increase the signal intensity around the beads in any case (Fig. 6 B, bottom right). Together, these results are consistent with the idea that RPTPα- and αv/β3-integrin–dependent reinforcement of integrin–cytoskeleton linkages is mediated by activation of SFK, in particular, activation of Fyn.


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)

Reinforcement requires RPTPα- and αv/β3-integrin–dependent activation of SFK. (A, top) RPTPα+/+ and RPTPα−/− cells were transfected with GFP-paxillin and were spread for 30 min. Large beads (5.9-μm diam) coated with FN were spun onto the cells and incubated for 30 min. The fraction of bound beads causing accumulation (fluorescence intensity >2× surrounding; inset) of paxillin was determined. Confocal stacks were resliced along the indicated line over the beads position (bottom of each panel), shown are overlays of GFP-paxillin fluorescence intensity in pseudo-colors and the differential interference contrast image. (middle) RPTPα+/+ and RPTPα−/− cells were pretreated with GPen. (bottom) RPTPα+/+ and RPTPα−/− cells were cotransfected with CSK and analyzed as described above. (B, top) Percentage of FN- or Con A–coated beads causing accumulation of paxillin. Results shown are the mean ± SD of three independent experiments. (bottom) RPTPα−/−wt cells (−/−wt) 30 min after application of beads. (bottom, right) RPTPα+/+ cells transfected with EGFP alone 30 min after application of beads. (C) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were spread for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. Accumulation was analyzed as described above. (left) Percentage of FN-coated beads causing accumulation of paxillin. (right) Representative micrographs of RPTPα−/− cells coexpressing Fyn (top), c-Src (bottom, left), c-Yes (bottom, right), and GFP-paxillin 30 min after application of FN-coated beads.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Reinforcement requires RPTPα- and αv/β3-integrin–dependent activation of SFK. (A, top) RPTPα+/+ and RPTPα−/− cells were transfected with GFP-paxillin and were spread for 30 min. Large beads (5.9-μm diam) coated with FN were spun onto the cells and incubated for 30 min. The fraction of bound beads causing accumulation (fluorescence intensity >2× surrounding; inset) of paxillin was determined. Confocal stacks were resliced along the indicated line over the beads position (bottom of each panel), shown are overlays of GFP-paxillin fluorescence intensity in pseudo-colors and the differential interference contrast image. (middle) RPTPα+/+ and RPTPα−/− cells were pretreated with GPen. (bottom) RPTPα+/+ and RPTPα−/− cells were cotransfected with CSK and analyzed as described above. (B, top) Percentage of FN- or Con A–coated beads causing accumulation of paxillin. Results shown are the mean ± SD of three independent experiments. (bottom) RPTPα−/−wt cells (−/−wt) 30 min after application of beads. (bottom, right) RPTPα+/+ cells transfected with EGFP alone 30 min after application of beads. (C) RPTPα+/+ (+/+) and RPTPα−/− cells (−/−) were spread for 30 min with or without cotransfection of wild-type Fyn, c-Src, or c-Yes and GFP-paxillin. Accumulation was analyzed as described above. (left) Percentage of FN-coated beads causing accumulation of paxillin. (right) Representative micrographs of RPTPα−/− cells coexpressing Fyn (top), c-Src (bottom, left), c-Yes (bottom, right), and GFP-paxillin 30 min after application of FN-coated beads.
Mentions: To further analyze the reinforcement process, we centrifuged onto cells (5 min/50 g) large beads (5.9-μm diam) coated with either FNIII7-10 or human VN. In contrast to the small beads used for the laser trap experiments, the large beads can cause the establishment of adhesion sites and, therefore, reinforcement by cellular contractions independent of laser trap restraint (Galbraith et al., 2002). Interestingly, 74% of the FNIII7-10–coated beads showed accumulation of GFP-paxillin to the bead periphery after 30 min on RPTPα+/+ cells (46% of VN-coated beads; unpublished data). In contrast, only 14% of the FNIII7-10–coated beads (7% of VN-coated beads; unpublished data) on RPTPα−/− cells showed redistribution of paxillin to the binding site (Fig. 6, A and B) . Next, we wanted to examine the function of SFK and αv/β3-integrins in the reinforcement process. Interestingly, pretreatment with GPen reduced the fraction of FNIII7-10–coated beads accumulating paxillin on RPTPα+/+ cells to 18%, but caused no significant reduction in the number of accumulating beads on the RPTPα−/− cells (11%; Fig. 6, A and B). Because the pretreatment with GPen prevented specific binding of VN-coated beads to the upper surface of RPTPα+/+ and RPTPα−/− cells (Fig. 5 A), there was no accumulation of paxillin around VN-coated beads in any case (unpublished data). Coexpression of CSK with paxillin decreased the number of FNIII7-10–coated beads, causing accumulation of paxillin to 16% in RPTPα+/+ cells, whereas there was no further reduction in RPTPα−/− cells (8%; Fig. 6, A and B). Consistent results could be obtained with VN-coated beads (8% for RPTPα+/+ cells and 5% for RPTPα−/− cells; unpublished data). We have shown earlier that RPTPα-dependent activation of Fyn is needed for the formation of focal complexes and contacts, suggesting that it might affect reinforcement. Indeed, coexpression of Fyn with GFP-paxillin led to a significant increase in the number of RPTPα−/− cells accumulating paxillin around beads (68%). In clear contrast, the coexpression of c-Src or c-Yes did not increase the number of accumulating beads in RPTPα−/− cells, but coexpression of c-Src reduced the number of cells responding to the beads in RPTPα+/+ cells to 34% (Fig. 6 C). Reintroduction of wild-type RPTPα into RPTPα−/− cells restored the ability to assemble paxillin at the binding site (Fig. 6 B). To confirm ligand specificity, we used Con A–coated beads on RPTPα+/+ cells, which showed accumulation of GFP-paxillin at the site of adhesion in only 7% of the beads, possibly due to unspecific activation of integrin receptors (Fig. 6 B). To exclude the possibility of a volume effect around the beads, we transfected RPTPα+/+ cells with EGFP alone, which did not increase the signal intensity around the beads in any case (Fig. 6 B, bottom right). Together, these results are consistent with the idea that RPTPα- and αv/β3-integrin–dependent reinforcement of integrin–cytoskeleton linkages is mediated by activation of SFK, in particular, activation of Fyn.

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