Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Apr 14;161(1):143-53.
doi: 10.1083/jcb.200211061. Epub 2003 Apr 7.

RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages

Gotz von Wichert  1 , Guoying Jiang, Ana Kostic, Kurt De Vos, Jan Sap, Michael P Sheetz
Affiliations
Free PMC article

RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages

Gotz von Wichert et al. J Cell Biol. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
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.
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 5.
Figure 5.
RPTPα deficiency does not affect integrin–ligand interactions, but leads to weak integrin–cytoskeleton linkages. (A) FN (left)- or VN (right)-coated beads (1-μm diam) bound specifically to the upper surface of RPTPα+/+ (+/+) and RPTPα−/− cells (−/−). Percentages of bound beads after 2 s were equalized with or without addition of GPen. Binding was compared with control beads coated with BSA. Results shown are the mean ± SD of three independent experiments. (B) Beads coated with FN (left) or VN (right) were placed on the upper surface of RPTPα+/+, RPTPα−/−, and RPTPα−/−wt cells, and the MSD was quantified after beads left the trap field (500 nm). Results shown are the mean ± SD of three independent experiments.
Figure 6.
Figure 6.
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.
Figure 7.
Figure 7.
Response to force requires expression of RPTPα. (A, top) Accumulation of GFP-paxillin in RPTPα+/+ cells (+/+) in serial micrographs of rearward moving beads coated with FN (1-μm diam) after placement on the upper surface and escape out of the trap. Beads position is indicated by an arrow. (bottom, left) Beads were placed on the upper surface and GFP-paxillin assembly was quantified without application of force in RPTPα+/+ cells. (bottom, right) Serial micrographs of RPTPα+/+ cells transfected with EGFP alone (left) and of GFP-paxillin transfected RPTPα+/+ cells after placement of Con A–coated beads (right). (B) Time-lapse micrographs of GFP-paxillin–expressing RPTPα−/− cells (−/−) after placement and escape out of the trap of FN-coated beads. (C) Time-lapse micrographs of rearward moving FN beads after placement and escape out of the trap on RPTPα−/−wt cells (−/−wt). (D) Model for the force-dependent assembly of focal complexes. First, upon formation of active lamellipodia, a complex of αv/β3-integrins and RPTPα is formed, localizing to the edge of the lamellipodium. Second, force application to αv/β3-integrins leads to RPTPα-dependent activation of SFK. Third, SFK-activation promotes the assembly and the reinforcement of focal complexes at early times. Finally, as focal complexes mature, SFK activity is also required for turnover of adhesion sites.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by 84 articles

See all "Cited by" articles

References

    1. Anton, E.S., J.A. Kreidberg, and P. Rakic. 1999. Distinct functions of alpha3 and alpha(v) integrin receptors in neuronal migration and laminar organization of the cerebral cortex. Neuron. 22:277–289. - PubMed
    1. Ardini, E., R. Agresti, E. Tagliabue, M. Greco, P. Aiello, L.T. Yang, S. Menard, and J. Sap. 2000. Expression of protein tyrosine phosphatase alpha (RPTPalpha) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo. Oncogene. 19:4979–4987. - PubMed
    1. Beningo, K.A., M. Dembo, I. Kaverina, J.V. Small, and Y.L. Wang. 2001. Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J. Cell Biol. 153:881–888. - PMC - PubMed
    1. Blystone, S.D., S.E. Slater, M.P. Williams, M.T. Crow, and E.J. Brown. 1999. A molecular mechanism of integrin crosstalk: αvβ3 suppression of calcium/calmodulin-dependent protein kinase II regulates α5β1 function. J. Cell Biol. 145:889–897. - PMC - PubMed
    1. Bockholt, S.M., and K. Burridge. 1995. An examination of focal adhesion formation and tyrosine phosphorylation in fibroblasts isolated from src-, fyn-, and yes- mice. Cell Adhes. Commun. 3:91–100. - PubMed

Publication types

MeSH terms