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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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Blockage of α4β1 paxillin associations interferes with shear resistance developed by wt α4. (A) JB4 cells expressing either wt α4 or α4(Y991A) were pretreated for 15 min with A7B7C7, a cell-permeable inhibitor of paxillin binding to the α4 tail, or with the control compound A6B6C6, both present at 5 μM. The shear resistance of carrier or compound-treated cells developed after 1-min adhesion to sVCAM-1 (2,220 sites/μm2) was determined as in Fig. 1. Results are mean ± range of two experimental fields. The experiments depicted are each representative of four independent tests. *, P < 0.001 (a two-tailed paired t test) for control compared with A7B7C7-treated cells at 0.5 dyn/cm2. (B) JB4 cells expressing wt α4 were transfected with either paxillin-specific or control luciferase siRNA. Total lysates of each group were immunoblotted with paxillin- or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 70 and 75% in paxillin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Paxillin silencing impairs resistance to detachment from sVCAM-1 developed by wt α4β1 but not α4(Y991A). The shear resistance of the indicated cells was determined as in A. Results are representative of three independent experiments. Error bars represent SD.
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fig3: Blockage of α4β1 paxillin associations interferes with shear resistance developed by wt α4. (A) JB4 cells expressing either wt α4 or α4(Y991A) were pretreated for 15 min with A7B7C7, a cell-permeable inhibitor of paxillin binding to the α4 tail, or with the control compound A6B6C6, both present at 5 μM. The shear resistance of carrier or compound-treated cells developed after 1-min adhesion to sVCAM-1 (2,220 sites/μm2) was determined as in Fig. 1. Results are mean ± range of two experimental fields. The experiments depicted are each representative of four independent tests. *, P < 0.001 (a two-tailed paired t test) for control compared with A7B7C7-treated cells at 0.5 dyn/cm2. (B) JB4 cells expressing wt α4 were transfected with either paxillin-specific or control luciferase siRNA. Total lysates of each group were immunoblotted with paxillin- or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 70 and 75% in paxillin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Paxillin silencing impairs resistance to detachment from sVCAM-1 developed by wt α4β1 but not α4(Y991A). The shear resistance of the indicated cells was determined as in A. Results are representative of three independent experiments. Error bars represent SD.

Mentions: The α4(Y991A) mutation blocks paxillin association with the α4 tail selectively (Liu et al., 1999). As an alternative test of the role of the α4–paxillin interaction, we exploited a recently identified small molecule inhibitor of this interaction. The compound, designated A7B7C7, blocks the α4–paxillin interaction and interferes with α4β1-dependent cell migration (Ambroise et al., 2002). This inhibitor, but not a control compound (A6B6C6), attenuated the shear resistance of wt α4β1–mediated Jurkat cell adhesion to VCAM-1 (Fig. 3 A, left) but had no effect on the residual shear resistance developed by the JB4-α4(Y991A) cells (Fig. 3 A, right). Adhesion mediated by the αLβ2–ICAM-1 interaction was also insensitive to the inhibitor (not depicted). Knocking down paxillin expression by up to 75% using transient short inhibitory RNA (siRNA) silencing (Fig. 3 B) resulted in reduced adhesiveness of wt α4β1–mediated Jurkat cell adhesion to VCAM-1 (Fig. 3 C), with no inhibition of adhesiveness mediated by the α4(Y991A) mutant (Fig. 3 C). Notably, LFA-1–dependent adhesion to ICAM-1 was also insensitive to identical paxillin silencing (not depicted). Thus, both genetic and pharmacological approaches indicate that the α4–paxillin interaction increases the resistance of α4β1–VCAM-1 contacts to detachment by disruptive shear stresses.


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)

Blockage of α4β1 paxillin associations interferes with shear resistance developed by wt α4. (A) JB4 cells expressing either wt α4 or α4(Y991A) were pretreated for 15 min with A7B7C7, a cell-permeable inhibitor of paxillin binding to the α4 tail, or with the control compound A6B6C6, both present at 5 μM. The shear resistance of carrier or compound-treated cells developed after 1-min adhesion to sVCAM-1 (2,220 sites/μm2) was determined as in Fig. 1. Results are mean ± range of two experimental fields. The experiments depicted are each representative of four independent tests. *, P < 0.001 (a two-tailed paired t test) for control compared with A7B7C7-treated cells at 0.5 dyn/cm2. (B) JB4 cells expressing wt α4 were transfected with either paxillin-specific or control luciferase siRNA. Total lysates of each group were immunoblotted with paxillin- or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 70 and 75% in paxillin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Paxillin silencing impairs resistance to detachment from sVCAM-1 developed by wt α4β1 but not α4(Y991A). The shear resistance of the indicated cells was determined as in A. Results are representative of three independent experiments. Error bars represent SD.
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fig3: Blockage of α4β1 paxillin associations interferes with shear resistance developed by wt α4. (A) JB4 cells expressing either wt α4 or α4(Y991A) were pretreated for 15 min with A7B7C7, a cell-permeable inhibitor of paxillin binding to the α4 tail, or with the control compound A6B6C6, both present at 5 μM. The shear resistance of carrier or compound-treated cells developed after 1-min adhesion to sVCAM-1 (2,220 sites/μm2) was determined as in Fig. 1. Results are mean ± range of two experimental fields. The experiments depicted are each representative of four independent tests. *, P < 0.001 (a two-tailed paired t test) for control compared with A7B7C7-treated cells at 0.5 dyn/cm2. (B) JB4 cells expressing wt α4 were transfected with either paxillin-specific or control luciferase siRNA. Total lysates of each group were immunoblotted with paxillin- or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 70 and 75% in paxillin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Paxillin silencing impairs resistance to detachment from sVCAM-1 developed by wt α4β1 but not α4(Y991A). The shear resistance of the indicated cells was determined as in A. Results are representative of three independent experiments. Error bars represent SD.
Mentions: The α4(Y991A) mutation blocks paxillin association with the α4 tail selectively (Liu et al., 1999). As an alternative test of the role of the α4–paxillin interaction, we exploited a recently identified small molecule inhibitor of this interaction. The compound, designated A7B7C7, blocks the α4–paxillin interaction and interferes with α4β1-dependent cell migration (Ambroise et al., 2002). This inhibitor, but not a control compound (A6B6C6), attenuated the shear resistance of wt α4β1–mediated Jurkat cell adhesion to VCAM-1 (Fig. 3 A, left) but had no effect on the residual shear resistance developed by the JB4-α4(Y991A) cells (Fig. 3 A, right). Adhesion mediated by the αLβ2–ICAM-1 interaction was also insensitive to the inhibitor (not depicted). Knocking down paxillin expression by up to 75% using transient short inhibitory RNA (siRNA) silencing (Fig. 3 B) resulted in reduced adhesiveness of wt α4β1–mediated Jurkat cell adhesion to VCAM-1 (Fig. 3 C), with no inhibition of adhesiveness mediated by the α4(Y991A) mutant (Fig. 3 C). Notably, LFA-1–dependent adhesion to ICAM-1 was also insensitive to identical paxillin silencing (not depicted). Thus, both genetic and pharmacological approaches indicate that the α4–paxillin interaction increases the resistance of α4β1–VCAM-1 contacts to detachment by disruptive shear stresses.

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