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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 the α4-cytoplasmic tail facilitates tethering mediated by α4β1 and α4β7 under shear flow without altering α4 distribution on microvilli. (A) Tethering (transient or followed by immediate arrest) of Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) to immobilized VCAM-1. The mean duration of transient tethers is shown in parenthesis above bars. Where indicated, cells were pretreated with 20 μM cytochalasin D (cyto D) or carrier (carr). Error bars represent SD. (B) Tethering under shear flow of Jurkat cells mediated by either WT or Y991A to distinct α4-integrin ligands. Tethers (transient or arrest) were determined under the indicated shear stresses on surfaces coated with either monomeric 7D VCAM-1 (sVCAM-1), dimeric 7D VCAM-1 (VCAM-1–Fc), or high density MadCAM-Fc. In each panel, the mean ± range of two experimental fields is depicted. All tethers to VCAM-1 were blocked in the presence of the α4-integrin mAb HP1/2 (not depicted). All tethers to MadCAM-1 were blocked by the anti-α4β7 antibody Act-I (not depicted). Results in A and B are representative of five and four independent experiments, respectively. (C) Surface distribution of wt α4 (WT) or the α4(Y991A) mutant on JB4 Jurkat cells monitored by immunoelectron microscopy. Insets show lower magnification images. The boxed areas depict the cellular areas enlarged. Prefixed cells were stained with the nonblocking α4-specific mAb B5G10. Washed cells were stained with rabbit anti–mouse Ig and 5 nm gold particle–conjugated goat anti–rabbit as described in Materials and methods. Gold particles are marked by arrowheads. Photomicrographs are representative of 20–30 cells.
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fig7: Paxillin association with the α4-cytoplasmic tail facilitates tethering mediated by α4β1 and α4β7 under shear flow without altering α4 distribution on microvilli. (A) Tethering (transient or followed by immediate arrest) of Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) to immobilized VCAM-1. The mean duration of transient tethers is shown in parenthesis above bars. Where indicated, cells were pretreated with 20 μM cytochalasin D (cyto D) or carrier (carr). Error bars represent SD. (B) Tethering under shear flow of Jurkat cells mediated by either WT or Y991A to distinct α4-integrin ligands. Tethers (transient or arrest) were determined under the indicated shear stresses on surfaces coated with either monomeric 7D VCAM-1 (sVCAM-1), dimeric 7D VCAM-1 (VCAM-1–Fc), or high density MadCAM-Fc. In each panel, the mean ± range of two experimental fields is depicted. All tethers to VCAM-1 were blocked in the presence of the α4-integrin mAb HP1/2 (not depicted). All tethers to MadCAM-1 were blocked by the anti-α4β7 antibody Act-I (not depicted). Results in A and B are representative of five and four independent experiments, respectively. (C) Surface distribution of wt α4 (WT) or the α4(Y991A) mutant on JB4 Jurkat cells monitored by immunoelectron microscopy. Insets show lower magnification images. The boxed areas depict the cellular areas enlarged. Prefixed cells were stained with the nonblocking α4-specific mAb B5G10. Washed cells were stained with rabbit anti–mouse Ig and 5 nm gold particle–conjugated goat anti–rabbit as described in Materials and methods. Gold particles are marked by arrowheads. Photomicrographs are representative of 20–30 cells.

Mentions: The α4β1 and α4β7 integrins mediate leukocyte capture under physiological shear flow (Alon et al., 1995; Berlin et al., 1995). Therefore, we compared the ability of mutant α4(Y991A) versus wt α4 to support α4β1-dependent T cell capture by monovalent and bivalent VCAM-1 under continuous shear flow. Consistent with its defective resistance to shear force, α4(Y991A)β1 mediated reduced cell capture and arrest on a large range of densities of either monovalent (sVCAM) or bivalent (VCAM-Fc) VCAM-1 (Fig. 7, A and B). This differential behavior was also manifested at different levels of shear stress that were tested (Fig. 7 B, first two panels). Notably, whereas disruption of the actin cytoskeleton by cytochalasin D resulted in marked inhibition of both cell capture and arrest mediated by wt α4β1, cytochalasin D had no effect on the residual adhesions mediated by the α4(Y991A)β1 mutant (Fig. 7 A). Interestingly, the duration of individual α4β1 tethers, which is a measure of integrin affinity to the ligand (Feigelson et al., 2001), was not altered upon the loss of paxillin binding (Fig. 7 A). Thus, for optimal cell capture under shear flow, the α4 tail of α4β1 requires associations with the intact actin cytoskeleton.


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 the α4-cytoplasmic tail facilitates tethering mediated by α4β1 and α4β7 under shear flow without altering α4 distribution on microvilli. (A) Tethering (transient or followed by immediate arrest) of Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) to immobilized VCAM-1. The mean duration of transient tethers is shown in parenthesis above bars. Where indicated, cells were pretreated with 20 μM cytochalasin D (cyto D) or carrier (carr). Error bars represent SD. (B) Tethering under shear flow of Jurkat cells mediated by either WT or Y991A to distinct α4-integrin ligands. Tethers (transient or arrest) were determined under the indicated shear stresses on surfaces coated with either monomeric 7D VCAM-1 (sVCAM-1), dimeric 7D VCAM-1 (VCAM-1–Fc), or high density MadCAM-Fc. In each panel, the mean ± range of two experimental fields is depicted. All tethers to VCAM-1 were blocked in the presence of the α4-integrin mAb HP1/2 (not depicted). All tethers to MadCAM-1 were blocked by the anti-α4β7 antibody Act-I (not depicted). Results in A and B are representative of five and four independent experiments, respectively. (C) Surface distribution of wt α4 (WT) or the α4(Y991A) mutant on JB4 Jurkat cells monitored by immunoelectron microscopy. Insets show lower magnification images. The boxed areas depict the cellular areas enlarged. Prefixed cells were stained with the nonblocking α4-specific mAb B5G10. Washed cells were stained with rabbit anti–mouse Ig and 5 nm gold particle–conjugated goat anti–rabbit as described in Materials and methods. Gold particles are marked by arrowheads. Photomicrographs are representative of 20–30 cells.
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fig7: Paxillin association with the α4-cytoplasmic tail facilitates tethering mediated by α4β1 and α4β7 under shear flow without altering α4 distribution on microvilli. (A) Tethering (transient or followed by immediate arrest) of Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) to immobilized VCAM-1. The mean duration of transient tethers is shown in parenthesis above bars. Where indicated, cells were pretreated with 20 μM cytochalasin D (cyto D) or carrier (carr). Error bars represent SD. (B) Tethering under shear flow of Jurkat cells mediated by either WT or Y991A to distinct α4-integrin ligands. Tethers (transient or arrest) were determined under the indicated shear stresses on surfaces coated with either monomeric 7D VCAM-1 (sVCAM-1), dimeric 7D VCAM-1 (VCAM-1–Fc), or high density MadCAM-Fc. In each panel, the mean ± range of two experimental fields is depicted. All tethers to VCAM-1 were blocked in the presence of the α4-integrin mAb HP1/2 (not depicted). All tethers to MadCAM-1 were blocked by the anti-α4β7 antibody Act-I (not depicted). Results in A and B are representative of five and four independent experiments, respectively. (C) Surface distribution of wt α4 (WT) or the α4(Y991A) mutant on JB4 Jurkat cells monitored by immunoelectron microscopy. Insets show lower magnification images. The boxed areas depict the cellular areas enlarged. Prefixed cells were stained with the nonblocking α4-specific mAb B5G10. Washed cells were stained with rabbit anti–mouse Ig and 5 nm gold particle–conjugated goat anti–rabbit as described in Materials and methods. Gold particles are marked by arrowheads. Photomicrographs are representative of 20–30 cells.
Mentions: The α4β1 and α4β7 integrins mediate leukocyte capture under physiological shear flow (Alon et al., 1995; Berlin et al., 1995). Therefore, we compared the ability of mutant α4(Y991A) versus wt α4 to support α4β1-dependent T cell capture by monovalent and bivalent VCAM-1 under continuous shear flow. Consistent with its defective resistance to shear force, α4(Y991A)β1 mediated reduced cell capture and arrest on a large range of densities of either monovalent (sVCAM) or bivalent (VCAM-Fc) VCAM-1 (Fig. 7, A and B). This differential behavior was also manifested at different levels of shear stress that were tested (Fig. 7 B, first two panels). Notably, whereas disruption of the actin cytoskeleton by cytochalasin D resulted in marked inhibition of both cell capture and arrest mediated by wt α4β1, cytochalasin D had no effect on the residual adhesions mediated by the α4(Y991A)β1 mutant (Fig. 7 A). Interestingly, the duration of individual α4β1 tethers, which is a measure of integrin affinity to the ligand (Feigelson et al., 2001), was not altered upon the loss of paxillin binding (Fig. 7 A). Thus, for optimal cell capture under shear flow, the α4 tail of α4β1 requires associations with the intact actin cytoskeleton.

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