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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 facilitates integrin anchorage to the cytoskeletal matrix. (A) Detergent removal of nonligated wt α4 monitored by FACS. Jurkat cells were reacted for 30 min at 4°C with FITC-conjugated anti-α4β1 mAb (HP1/2) or isotype-matched control (dotted line). Cells were then incubated at RT in detergent-free buffer (black, −NP-40) or in buffer containing 0.05% NP-40 (gray, +NP-40). The fraction of α4-bound mAb remaining after detergent treatment assessed by flow cytometry is shown relative to originally bound α4 mAb. (B) The fraction of mAb-bound wt or α4(Y991A) resistant to detergent-induced removal was compared before (white bars) and after ligation of the mAb by secondary antibody (black bars). Results are a mean of three independent experiments. Error bars represent SD.
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fig4: Paxillin association with α4 facilitates integrin anchorage to the cytoskeletal matrix. (A) Detergent removal of nonligated wt α4 monitored by FACS. Jurkat cells were reacted for 30 min at 4°C with FITC-conjugated anti-α4β1 mAb (HP1/2) or isotype-matched control (dotted line). Cells were then incubated at RT in detergent-free buffer (black, −NP-40) or in buffer containing 0.05% NP-40 (gray, +NP-40). The fraction of α4-bound mAb remaining after detergent treatment assessed by flow cytometry is shown relative to originally bound α4 mAb. (B) The fraction of mAb-bound wt or α4(Y991A) resistant to detergent-induced removal was compared before (white bars) and after ligation of the mAb by secondary antibody (black bars). Results are a mean of three independent experiments. Error bars represent SD.

Mentions: Paxillin binds a number of actin-binding proteins such as talin and vinculin (Brown and Turner, 2004) and does so at sites distinct from the α4-binding site (Liu and Ginsberg, 2000). We next quantified the fraction of detergent-resistant wt α4 or α4(Y991A) retained on NP-40–solubilized cells using fluorescence-tagged integrin-bound α4 mAb (Fig. 4 A). Retention of intact wt and α4(Y991A) was similar and low (20% of the total surface α4; Fig. 4 A). However, the addition of anti–mouse Ig to cluster the mAb-bound wt α4 markedly increased the association of α4β1 surface integrin with the detergent-insoluble cytoskeleton (Fig. 4 B). In contrast, the same treatment produced a negligible increase in the cytoskeletal association of α4(Y991A)β1 (Fig. 4 B). Thus, the α4(Y991A) mutant fails to anchor properly to the actin cytoskeleton in Jurkat T cells.


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 facilitates integrin anchorage to the cytoskeletal matrix. (A) Detergent removal of nonligated wt α4 monitored by FACS. Jurkat cells were reacted for 30 min at 4°C with FITC-conjugated anti-α4β1 mAb (HP1/2) or isotype-matched control (dotted line). Cells were then incubated at RT in detergent-free buffer (black, −NP-40) or in buffer containing 0.05% NP-40 (gray, +NP-40). The fraction of α4-bound mAb remaining after detergent treatment assessed by flow cytometry is shown relative to originally bound α4 mAb. (B) The fraction of mAb-bound wt or α4(Y991A) resistant to detergent-induced removal was compared before (white bars) and after ligation of the mAb by secondary antibody (black bars). Results are a mean of three independent experiments. Error bars represent SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171310&req=5

fig4: Paxillin association with α4 facilitates integrin anchorage to the cytoskeletal matrix. (A) Detergent removal of nonligated wt α4 monitored by FACS. Jurkat cells were reacted for 30 min at 4°C with FITC-conjugated anti-α4β1 mAb (HP1/2) or isotype-matched control (dotted line). Cells were then incubated at RT in detergent-free buffer (black, −NP-40) or in buffer containing 0.05% NP-40 (gray, +NP-40). The fraction of α4-bound mAb remaining after detergent treatment assessed by flow cytometry is shown relative to originally bound α4 mAb. (B) The fraction of mAb-bound wt or α4(Y991A) resistant to detergent-induced removal was compared before (white bars) and after ligation of the mAb by secondary antibody (black bars). Results are a mean of three independent experiments. Error bars represent SD.
Mentions: Paxillin binds a number of actin-binding proteins such as talin and vinculin (Brown and Turner, 2004) and does so at sites distinct from the α4-binding site (Liu and Ginsberg, 2000). We next quantified the fraction of detergent-resistant wt α4 or α4(Y991A) retained on NP-40–solubilized cells using fluorescence-tagged integrin-bound α4 mAb (Fig. 4 A). Retention of intact wt and α4(Y991A) was similar and low (20% of the total surface α4; Fig. 4 A). However, the addition of anti–mouse Ig to cluster the mAb-bound wt α4 markedly increased the association of α4β1 surface integrin with the detergent-insoluble cytoskeleton (Fig. 4 B). In contrast, the same treatment produced a negligible increase in the cytoskeletal association of α4(Y991A)β1 (Fig. 4 B). Thus, the α4(Y991A) mutant fails to anchor properly to the actin cytoskeleton in Jurkat T cells.

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