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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 distributes normally before and during early cell spreading on VCAM-1 in shear-free conditions. (A) wt α4 or α4(Y991A) is evenly distributed on the surface of JB4 cells. Confocal immunostaining of α4 on the surface of prefixed WT or Y991A cells using the nonblocking B5G10 mAb. Three representative cells are shown for each cell type. (B) Live imaging of wt α4 or α4(Y991A) during short cellular contacts with VCAM-1. JB4 cells expressing wt or mutant α4 were prelabeled with AlexaFluor488-conjugated B5G10 mAb and settled for 1 min on VCAM-1. wt or mutant α4 were each imaged on cells that had spread on sVCAM-1 for 1 min (shear free) and were then subjected to 10 s of shear stress at 2 dyn/cm2. Cell morphology was monitored in differential interface microscopy (DIC). The degree of patching was calculated by Image J analysis and was defined as having at least one region with a B5G10 staining mean intensity threefold higher than another region on the same cell. Note that shear stress on its own did not trigger wt α4 redistribution. Shear direction is depicted by the arrow.
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fig2: The α4(Y991A)β1 mutant distributes normally before and during early cell spreading on VCAM-1 in shear-free conditions. (A) wt α4 or α4(Y991A) is evenly distributed on the surface of JB4 cells. Confocal immunostaining of α4 on the surface of prefixed WT or Y991A cells using the nonblocking B5G10 mAb. Three representative cells are shown for each cell type. (B) Live imaging of wt α4 or α4(Y991A) during short cellular contacts with VCAM-1. JB4 cells expressing wt or mutant α4 were prelabeled with AlexaFluor488-conjugated B5G10 mAb and settled for 1 min on VCAM-1. wt or mutant α4 were each imaged on cells that had spread on sVCAM-1 for 1 min (shear free) and were then subjected to 10 s of shear stress at 2 dyn/cm2. Cell morphology was monitored in differential interface microscopy (DIC). The degree of patching was calculated by Image J analysis and was defined as having at least one region with a B5G10 staining mean intensity threefold higher than another region on the same cell. Note that shear stress on its own did not trigger wt α4 redistribution. Shear direction is depicted by the arrow.

Mentions: Notably, preformed clustering of wt and mutant α4 subunits on JB4 cells was essentially identical (Fig. 2 A). Real time imaging of JB4 cells that adhered on VCAM-1 also showed identical cell spreading as well as the distribution of both mutant and wt α4 during 1-min cellular contacts before shear application (WT: n = 44, 16% round, 54% polarized with uniform α4, 30% polarized with patched α4; Y911A: n = 27, 18% round, 52% polarized with uniform α4, 30% polarized with patched α4; Fig. 2 B). Notably, the strength of resistance to detachment developed by wt α4 did not correlate with the degree of patching (Fig. 2 B) in contrast to reports on LFA-1–dependent systems (Constantin et al., 2000; Kim et al., 2004). Thus, a mutation of the α4 tail defective in paxillin binding prevents α4β1-mediated resistance to shear-induced cell detachment independent of cell spreading and α4 patching on VCAM-1.


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 distributes normally before and during early cell spreading on VCAM-1 in shear-free conditions. (A) wt α4 or α4(Y991A) is evenly distributed on the surface of JB4 cells. Confocal immunostaining of α4 on the surface of prefixed WT or Y991A cells using the nonblocking B5G10 mAb. Three representative cells are shown for each cell type. (B) Live imaging of wt α4 or α4(Y991A) during short cellular contacts with VCAM-1. JB4 cells expressing wt or mutant α4 were prelabeled with AlexaFluor488-conjugated B5G10 mAb and settled for 1 min on VCAM-1. wt or mutant α4 were each imaged on cells that had spread on sVCAM-1 for 1 min (shear free) and were then subjected to 10 s of shear stress at 2 dyn/cm2. Cell morphology was monitored in differential interface microscopy (DIC). The degree of patching was calculated by Image J analysis and was defined as having at least one region with a B5G10 staining mean intensity threefold higher than another region on the same cell. Note that shear stress on its own did not trigger wt α4 redistribution. Shear direction is depicted by the arrow.
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Related In: Results  -  Collection

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fig2: The α4(Y991A)β1 mutant distributes normally before and during early cell spreading on VCAM-1 in shear-free conditions. (A) wt α4 or α4(Y991A) is evenly distributed on the surface of JB4 cells. Confocal immunostaining of α4 on the surface of prefixed WT or Y991A cells using the nonblocking B5G10 mAb. Three representative cells are shown for each cell type. (B) Live imaging of wt α4 or α4(Y991A) during short cellular contacts with VCAM-1. JB4 cells expressing wt or mutant α4 were prelabeled with AlexaFluor488-conjugated B5G10 mAb and settled for 1 min on VCAM-1. wt or mutant α4 were each imaged on cells that had spread on sVCAM-1 for 1 min (shear free) and were then subjected to 10 s of shear stress at 2 dyn/cm2. Cell morphology was monitored in differential interface microscopy (DIC). The degree of patching was calculated by Image J analysis and was defined as having at least one region with a B5G10 staining mean intensity threefold higher than another region on the same cell. Note that shear stress on its own did not trigger wt α4 redistribution. Shear direction is depicted by the arrow.
Mentions: Notably, preformed clustering of wt and mutant α4 subunits on JB4 cells was essentially identical (Fig. 2 A). Real time imaging of JB4 cells that adhered on VCAM-1 also showed identical cell spreading as well as the distribution of both mutant and wt α4 during 1-min cellular contacts before shear application (WT: n = 44, 16% round, 54% polarized with uniform α4, 30% polarized with patched α4; Y911A: n = 27, 18% round, 52% polarized with uniform α4, 30% polarized with patched α4; Fig. 2 B). Notably, the strength of resistance to detachment developed by wt α4 did not correlate with the degree of patching (Fig. 2 B) in contrast to reports on LFA-1–dependent systems (Constantin et al., 2000; Kim et al., 2004). Thus, a mutation of the α4 tail defective in paxillin binding prevents α4β1-mediated resistance to shear-induced cell detachment independent of cell spreading and α4 patching on VCAM-1.

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