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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 responds to phorbol ester and SDF-1 inside-out signals but develops poor adhesiveness in stimulated T cells under shear flow. Adhesion of JB4 cells expressing wt α4 or α4(Y991A) mutant to sVCAM-1 (2,960 sites/μm2) left intact (−) or stimulated by 1 min PMA pretreatment or by cell encounter with SDF-1α coimmobilized at 2 μg/ml. (A) Resistance to detachment after 1 min of static contact analyzed as in Fig. 1. Values are mean ± range of two experimental fields. (B) Capture and arrest under continuous shear flow. Frequency of tethers and their categories were determined as in Fig. 7. The experiments in A and B are each representative of four independent tests.
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fig10: The α4(Y991A)β1 mutant responds to phorbol ester and SDF-1 inside-out signals but develops poor adhesiveness in stimulated T cells under shear flow. Adhesion of JB4 cells expressing wt α4 or α4(Y991A) mutant to sVCAM-1 (2,960 sites/μm2) left intact (−) or stimulated by 1 min PMA pretreatment or by cell encounter with SDF-1α coimmobilized at 2 μg/ml. (A) Resistance to detachment after 1 min of static contact analyzed as in Fig. 1. Values are mean ± range of two experimental fields. (B) Capture and arrest under continuous shear flow. Frequency of tethers and their categories were determined as in Fig. 7. The experiments in A and B are each representative of four independent tests.

Mentions: Cellular stimulation by various agonists increases integrin adhesiveness in various contexts (Hynes, 2002). We next examined the effects of two prototypic agonists, PMA, a direct agonist of diacyl glycerol–dependent PKCs, and the chemokine SDF-1 (CXCL12) on mutant and wt α4. Exposure of JB4-α4(Y991A) cells to soluble PMA or to immobilized SDF-1 resulted in enhanced resistance to detachment from VCAM-1–bearing surfaces (Fig. 10 A), although the overall adhesion strength developed by the α4(Y991A) mutant was lower than that developed by the intact integrin. Further analysis of adhesive tethers formed on VCAM-1 at subsecond contacts also indicated that the ability of initial α4(Y991A)β1-dependent Jurkat tethers to convert to immediate firm arrests was enhanced by PMA (Fig. 10 B). Likewise, JB4-α4(Y991A) cells efficiently responded to in situ subsecond signals from SDF-1 with a twofold elevated frequency of α4β1-dependent tethers on VCAM-1 (Fig. 10 B, right). However, overall SDF-1–stimulated tethers mediated by α4(Y991A) cells remained lower than wt α4–mediated tethers. Thus, although the mutant α4 underwent robust activation in response to both chemokine and PMA inside-out signals, its impaired cytoskeletal associations resulted in overall reduced adhesion to VCAM-1 under shear stress.


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 responds to phorbol ester and SDF-1 inside-out signals but develops poor adhesiveness in stimulated T cells under shear flow. Adhesion of JB4 cells expressing wt α4 or α4(Y991A) mutant to sVCAM-1 (2,960 sites/μm2) left intact (−) or stimulated by 1 min PMA pretreatment or by cell encounter with SDF-1α coimmobilized at 2 μg/ml. (A) Resistance to detachment after 1 min of static contact analyzed as in Fig. 1. Values are mean ± range of two experimental fields. (B) Capture and arrest under continuous shear flow. Frequency of tethers and their categories were determined as in Fig. 7. The experiments in A and B are each representative of four independent tests.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171310&req=5

fig10: The α4(Y991A)β1 mutant responds to phorbol ester and SDF-1 inside-out signals but develops poor adhesiveness in stimulated T cells under shear flow. Adhesion of JB4 cells expressing wt α4 or α4(Y991A) mutant to sVCAM-1 (2,960 sites/μm2) left intact (−) or stimulated by 1 min PMA pretreatment or by cell encounter with SDF-1α coimmobilized at 2 μg/ml. (A) Resistance to detachment after 1 min of static contact analyzed as in Fig. 1. Values are mean ± range of two experimental fields. (B) Capture and arrest under continuous shear flow. Frequency of tethers and their categories were determined as in Fig. 7. The experiments in A and B are each representative of four independent tests.
Mentions: Cellular stimulation by various agonists increases integrin adhesiveness in various contexts (Hynes, 2002). We next examined the effects of two prototypic agonists, PMA, a direct agonist of diacyl glycerol–dependent PKCs, and the chemokine SDF-1 (CXCL12) on mutant and wt α4. Exposure of JB4-α4(Y991A) cells to soluble PMA or to immobilized SDF-1 resulted in enhanced resistance to detachment from VCAM-1–bearing surfaces (Fig. 10 A), although the overall adhesion strength developed by the α4(Y991A) mutant was lower than that developed by the intact integrin. Further analysis of adhesive tethers formed on VCAM-1 at subsecond contacts also indicated that the ability of initial α4(Y991A)β1-dependent Jurkat tethers to convert to immediate firm arrests was enhanced by PMA (Fig. 10 B). Likewise, JB4-α4(Y991A) cells efficiently responded to in situ subsecond signals from SDF-1 with a twofold elevated frequency of α4β1-dependent tethers on VCAM-1 (Fig. 10 B, right). However, overall SDF-1–stimulated tethers mediated by α4(Y991A) cells remained lower than wt α4–mediated tethers. Thus, although the mutant α4 underwent robust activation in response to both chemokine and PMA inside-out signals, its impaired cytoskeletal associations resulted in overall reduced adhesion to VCAM-1 under shear stress.

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