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Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.

Alon R, Feigelson SW, Manevich E, Rose DM, Schmitz J, Overby DR, Winter E, Grabovsky V, Shinder V, Matthews BD, Sokolovsky-Eisenberg M, Ingber DE, Benoit M, Ginsberg MH - J. Cell Biol. (2005)

Bottom Line: The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations.In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin.The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. ronen.alon@weizmann.ac.il

ABSTRACT
The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

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Paxillin association with α4 integrins stabilizes adhesive tethers to immobilized α4-specific mAbs independent of ligand-induced rearrangements. (A) Dose-dependent induction of the 15/7 epitope by the α4β1-specific ligand Bio1211 on wt α4 or α4(Y991A)–expressing Jurkat cells. (B) Reduced tethering and firm adhesion of the α4(Y991A) mutant to immobilized α4 mAb (HP1/2) under shear flow. Frequency of tethers and their categories were determined as in Fig. 7. (C) Strength of adhesion developed by JB4 expressing either wt or α4(Y991A) settled for 1 min on low or high density mAb. Experiments in A and B are each representative of three independent experiments. Error bars represent SD.
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fig9: Paxillin association with α4 integrins stabilizes adhesive tethers to immobilized α4-specific mAbs independent of ligand-induced rearrangements. (A) Dose-dependent induction of the 15/7 epitope by the α4β1-specific ligand Bio1211 on wt α4 or α4(Y991A)–expressing Jurkat cells. (B) Reduced tethering and firm adhesion of the α4(Y991A) mutant to immobilized α4 mAb (HP1/2) under shear flow. Frequency of tethers and their categories were determined as in Fig. 7. (C) Strength of adhesion developed by JB4 expressing either wt or α4(Y991A) settled for 1 min on low or high density mAb. Experiments in A and B are each representative of three independent experiments. Error bars represent SD.

Mentions: The aforementioned data suggest that paxillin binding to α4 is required for mechanical stabilization of cell attachments rather than for cytoplasmic induction of high affinity integrin conformations. Ligand binding to integrins can induce conformational changes in the integrin, resulting in high affinity conformations (Du et al., 1991; Shimaoka et al., 2002). Therefore, we considered the possibility that the reduced tether formation by the α4(Y991A) mutant could reflect defective, instantaneous ligand-induced conformational changes in the integrin under shear stress. We first verified that the α4β1 ligand Bio1211 provoked similar conformational changes in α4β1 and α4(Y991A)β1 under shear-free conditions, as indicated by the identical induction of the β1 ligand–induced binding site reporter 15/7 epitope by increasing doses of the monovalent α4β1-specific ligand Bio1211 (Fig. 9 A; Lin et al., 1999). We next tested the intrinsic attachment efficacy of either wt α4 or the α4Y991A mutant to surface-immobilized α4 mAb in the absence of ligand occupancy of the integrin. Notably, the HP1/2 mAb binding to α4 integrins is not sensitive to their affinity to or rearrangement by native ligands (Feigelson et al., 2001) and, thus, should be insensitive to intrinsic or ligand-induced affinity changes under shear stress. Notably, in the presence of shear flow, the α4(Y991A) mutant formed adhesive tethers to immobilized HP1/2 mAb much less efficiently than wt α4 (Fig. 9 B), as was observed for VCAM-1 (Fig. 1). In addition, adhesive contacts generated by the α4(Y991A) mutant after 1 min of static contact also exhibited poor resistance to detachment by increasing shear forces relative to wt α4–mediated contacts (Fig. 9 C). Thus, paxillin association with the α4-integrin tail enhances the ability of the integrin subunit to generate resistance to detachment forces independently of ligand-induced conformational rearrangements under shear stress conditions.


Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.

Alon R, Feigelson SW, Manevich E, Rose DM, Schmitz J, Overby DR, Winter E, Grabovsky V, Shinder V, Matthews BD, Sokolovsky-Eisenberg M, Ingber DE, Benoit M, Ginsberg MH - J. Cell Biol. (2005)

Paxillin association with α4 integrins stabilizes adhesive tethers to immobilized α4-specific mAbs independent of ligand-induced rearrangements. (A) Dose-dependent induction of the 15/7 epitope by the α4β1-specific ligand Bio1211 on wt α4 or α4(Y991A)–expressing Jurkat cells. (B) Reduced tethering and firm adhesion of the α4(Y991A) mutant to immobilized α4 mAb (HP1/2) under shear flow. Frequency of tethers and their categories were determined as in Fig. 7. (C) Strength of adhesion developed by JB4 expressing either wt or α4(Y991A) settled for 1 min on low or high density mAb. Experiments in A and B are each representative of three independent experiments. Error bars represent SD.
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fig9: Paxillin association with α4 integrins stabilizes adhesive tethers to immobilized α4-specific mAbs independent of ligand-induced rearrangements. (A) Dose-dependent induction of the 15/7 epitope by the α4β1-specific ligand Bio1211 on wt α4 or α4(Y991A)–expressing Jurkat cells. (B) Reduced tethering and firm adhesion of the α4(Y991A) mutant to immobilized α4 mAb (HP1/2) under shear flow. Frequency of tethers and their categories were determined as in Fig. 7. (C) Strength of adhesion developed by JB4 expressing either wt or α4(Y991A) settled for 1 min on low or high density mAb. Experiments in A and B are each representative of three independent experiments. Error bars represent SD.
Mentions: The aforementioned data suggest that paxillin binding to α4 is required for mechanical stabilization of cell attachments rather than for cytoplasmic induction of high affinity integrin conformations. Ligand binding to integrins can induce conformational changes in the integrin, resulting in high affinity conformations (Du et al., 1991; Shimaoka et al., 2002). Therefore, we considered the possibility that the reduced tether formation by the α4(Y991A) mutant could reflect defective, instantaneous ligand-induced conformational changes in the integrin under shear stress. We first verified that the α4β1 ligand Bio1211 provoked similar conformational changes in α4β1 and α4(Y991A)β1 under shear-free conditions, as indicated by the identical induction of the β1 ligand–induced binding site reporter 15/7 epitope by increasing doses of the monovalent α4β1-specific ligand Bio1211 (Fig. 9 A; Lin et al., 1999). We next tested the intrinsic attachment efficacy of either wt α4 or the α4Y991A mutant to surface-immobilized α4 mAb in the absence of ligand occupancy of the integrin. Notably, the HP1/2 mAb binding to α4 integrins is not sensitive to their affinity to or rearrangement by native ligands (Feigelson et al., 2001) and, thus, should be insensitive to intrinsic or ligand-induced affinity changes under shear stress. Notably, in the presence of shear flow, the α4(Y991A) mutant formed adhesive tethers to immobilized HP1/2 mAb much less efficiently than wt α4 (Fig. 9 B), as was observed for VCAM-1 (Fig. 1). In addition, adhesive contacts generated by the α4(Y991A) mutant after 1 min of static contact also exhibited poor resistance to detachment by increasing shear forces relative to wt α4–mediated contacts (Fig. 9 C). Thus, paxillin association with the α4-integrin tail enhances the ability of the integrin subunit to generate resistance to detachment forces independently of ligand-induced conformational rearrangements under shear stress conditions.

Bottom Line: The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations.In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin.The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. ronen.alon@weizmann.ac.il

ABSTRACT
The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

Show MeSH
Related in: MedlinePlus