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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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The α4(Y991A)β1 mutant exhibits normal avidity under shear-free conditions but develops lower bond stiffness under applied force. (A) Binding of either wt or Y991A α4β1-expressing cells to M-280 protein A Dynabeads coated with 2D VCAM-1–Fc. Relative bead binding was determined by side scattering analysis. Bead binding in the presence of 1 μg/ml of the α4β1-specific blocker Bio1211 is shown in gray squares. Results are representative of three independent experiments. (B, top) Representative bead displacement measured from an α4(Y991A)-expressing cell (open circles) and a wt α4–expressing cell (closed circles) during a 500-ms force pulse of ∼100 pN. (bottom) Electromagnetic current waveform corresponding to the displacement response. (C) VCAM-1–coated magnetic bead displacement in response to magnetic force pulse. VCAM-1 beads bound on the surface of JB4 cells expressing wt or α4(Y991A) as well as on cytochalasin D–treated JB4 cells expressing wt α4 were exposed for 0.5 s to the force pulse as described in the supplemental Materials and methods and Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503155/DC1). For each experimental group, 8–10 cells were analyzed, and results are the mean ± SD (error bars) of all displacement curves. All samples were confirmed by side scattering analysis to bind a similar number of VCAM-1 beads. A two-tailed unpaired t test for mean bead displacements on wt and α4(Y991A)-expressing JB4 cells yielded P < 0.06. One representative experiment of three.
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fig6: The α4(Y991A)β1 mutant exhibits normal avidity under shear-free conditions but develops lower bond stiffness under applied force. (A) Binding of either wt or Y991A α4β1-expressing cells to M-280 protein A Dynabeads coated with 2D VCAM-1–Fc. Relative bead binding was determined by side scattering analysis. Bead binding in the presence of 1 μg/ml of the α4β1-specific blocker Bio1211 is shown in gray squares. Results are representative of three independent experiments. (B, top) Representative bead displacement measured from an α4(Y991A)-expressing cell (open circles) and a wt α4–expressing cell (closed circles) during a 500-ms force pulse of ∼100 pN. (bottom) Electromagnetic current waveform corresponding to the displacement response. (C) VCAM-1–coated magnetic bead displacement in response to magnetic force pulse. VCAM-1 beads bound on the surface of JB4 cells expressing wt or α4(Y991A) as well as on cytochalasin D–treated JB4 cells expressing wt α4 were exposed for 0.5 s to the force pulse as described in the supplemental Materials and methods and Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503155/DC1). For each experimental group, 8–10 cells were analyzed, and results are the mean ± SD (error bars) of all displacement curves. All samples were confirmed by side scattering analysis to bind a similar number of VCAM-1 beads. A two-tailed unpaired t test for mean bead displacements on wt and α4(Y991A)-expressing JB4 cells yielded P < 0.06. One representative experiment of three.

Mentions: Although the affinity of integrin α4(Y991A)β1 to soluble VCAM-1–Fc is retained (Rose et al., 2003), we considered that Jurkat cells expressing the α4(Y991A)β1 mutant might fail to develop shear resistant adhesion as a result of reduced avidity for surface-bound VCAM-1. Comparing wt α4β1 with α4(Y991A)β1 adhesiveness to VCAM-1–coated beads in the absence of applied shear stress, we found that JB4-wt and JB4-α4(Y991A) cells bound identically to magnetic beads coated with increasing site densities of VCAM-1–Fc (Fig. 6 A), which is supportive of the normal adhesion of α4(Y991A)β1-expressing cells under static conditions. Nevertheless, when VCAM-1–coated beads that bound to JB4-wt cells were exposed to abrupt mechanical stress, these beads were displaced significantly less than beads prebound to JB4-α4(Y991A) cells (Fig. 6 B and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200503155/DC1). α4 resistance to displacement required an intact actin cytoskeleton, as JB4-wt cells pretreated with the F-actin–severing drug cytochalasin D exhibited even greater displacement in response to abrupt magnetic stress (Fig. 6 C). These findings, together with the shear-based detachment assays (Fig. 1), collectively suggest that paxillin association with the α4β1 heterodimer and an intact actin cytoskeleton are both required for ligand-occupied α4β1 to develop stress-resistant adhesive bonds.


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)

The α4(Y991A)β1 mutant exhibits normal avidity under shear-free conditions but develops lower bond stiffness under applied force. (A) Binding of either wt or Y991A α4β1-expressing cells to M-280 protein A Dynabeads coated with 2D VCAM-1–Fc. Relative bead binding was determined by side scattering analysis. Bead binding in the presence of 1 μg/ml of the α4β1-specific blocker Bio1211 is shown in gray squares. Results are representative of three independent experiments. (B, top) Representative bead displacement measured from an α4(Y991A)-expressing cell (open circles) and a wt α4–expressing cell (closed circles) during a 500-ms force pulse of ∼100 pN. (bottom) Electromagnetic current waveform corresponding to the displacement response. (C) VCAM-1–coated magnetic bead displacement in response to magnetic force pulse. VCAM-1 beads bound on the surface of JB4 cells expressing wt or α4(Y991A) as well as on cytochalasin D–treated JB4 cells expressing wt α4 were exposed for 0.5 s to the force pulse as described in the supplemental Materials and methods and Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503155/DC1). For each experimental group, 8–10 cells were analyzed, and results are the mean ± SD (error bars) of all displacement curves. All samples were confirmed by side scattering analysis to bind a similar number of VCAM-1 beads. A two-tailed unpaired t test for mean bead displacements on wt and α4(Y991A)-expressing JB4 cells yielded P < 0.06. One representative experiment of three.
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fig6: The α4(Y991A)β1 mutant exhibits normal avidity under shear-free conditions but develops lower bond stiffness under applied force. (A) Binding of either wt or Y991A α4β1-expressing cells to M-280 protein A Dynabeads coated with 2D VCAM-1–Fc. Relative bead binding was determined by side scattering analysis. Bead binding in the presence of 1 μg/ml of the α4β1-specific blocker Bio1211 is shown in gray squares. Results are representative of three independent experiments. (B, top) Representative bead displacement measured from an α4(Y991A)-expressing cell (open circles) and a wt α4–expressing cell (closed circles) during a 500-ms force pulse of ∼100 pN. (bottom) Electromagnetic current waveform corresponding to the displacement response. (C) VCAM-1–coated magnetic bead displacement in response to magnetic force pulse. VCAM-1 beads bound on the surface of JB4 cells expressing wt or α4(Y991A) as well as on cytochalasin D–treated JB4 cells expressing wt α4 were exposed for 0.5 s to the force pulse as described in the supplemental Materials and methods and Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503155/DC1). For each experimental group, 8–10 cells were analyzed, and results are the mean ± SD (error bars) of all displacement curves. All samples were confirmed by side scattering analysis to bind a similar number of VCAM-1 beads. A two-tailed unpaired t test for mean bead displacements on wt and α4(Y991A)-expressing JB4 cells yielded P < 0.06. One representative experiment of three.
Mentions: Although the affinity of integrin α4(Y991A)β1 to soluble VCAM-1–Fc is retained (Rose et al., 2003), we considered that Jurkat cells expressing the α4(Y991A)β1 mutant might fail to develop shear resistant adhesion as a result of reduced avidity for surface-bound VCAM-1. Comparing wt α4β1 with α4(Y991A)β1 adhesiveness to VCAM-1–coated beads in the absence of applied shear stress, we found that JB4-wt and JB4-α4(Y991A) cells bound identically to magnetic beads coated with increasing site densities of VCAM-1–Fc (Fig. 6 A), which is supportive of the normal adhesion of α4(Y991A)β1-expressing cells under static conditions. Nevertheless, when VCAM-1–coated beads that bound to JB4-wt cells were exposed to abrupt mechanical stress, these beads were displaced significantly less than beads prebound to JB4-α4(Y991A) cells (Fig. 6 B and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200503155/DC1). α4 resistance to displacement required an intact actin cytoskeleton, as JB4-wt cells pretreated with the F-actin–severing drug cytochalasin D exhibited even greater displacement in response to abrupt magnetic stress (Fig. 6 C). These findings, together with the shear-based detachment assays (Fig. 1), collectively suggest that paxillin association with the α4β1 heterodimer and an intact actin cytoskeleton are both required for ligand-occupied α4β1 to develop stress-resistant adhesive bonds.

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