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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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α4(Y991A)β1 fails to stabilize bonds ruptured by an AFM probe. (A) Schematic representation of the experimental system. JB4 cells were coupled to an AFM cantilever tip via an anti-CD43 mAb. VCAM-Fc was immobilized onto the substrate as in previous figures. (B) Representative AFM force–displacement curves acquired with wt α4–expressing JB4 cells (top) or α4(Y991A)-expressing cells (middle) approaching the VCAM-1–Fc-bearing substrate. A force–displacement curve of wt α4–expressing JB4 approaching a control substrate devoid of VCAM-1 is indicated in the bottom curve. (C) Force histograms of α4β1–VCAM-1 unbinding forces measured under a fixed loading rate of 0.33 nN/s. The number of productive adhesive interactions and their unbinding force distribution are depicted. Background binding is depicted by the dashed line. The mean unbinding force (UF) values of 10 independent experiments are indicated near each histogram. Pulling velocity was 3 μm/s, and the cell–substrate contact time was 0.5 s. A representative result of 10 independent experiments is depicted.
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fig8: α4(Y991A)β1 fails to stabilize bonds ruptured by an AFM probe. (A) Schematic representation of the experimental system. JB4 cells were coupled to an AFM cantilever tip via an anti-CD43 mAb. VCAM-Fc was immobilized onto the substrate as in previous figures. (B) Representative AFM force–displacement curves acquired with wt α4–expressing JB4 cells (top) or α4(Y991A)-expressing cells (middle) approaching the VCAM-1–Fc-bearing substrate. A force–displacement curve of wt α4–expressing JB4 approaching a control substrate devoid of VCAM-1 is indicated in the bottom curve. (C) Force histograms of α4β1–VCAM-1 unbinding forces measured under a fixed loading rate of 0.33 nN/s. The number of productive adhesive interactions and their unbinding force distribution are depicted. Background binding is depicted by the dashed line. The mean unbinding force (UF) values of 10 independent experiments are indicated near each histogram. Pulling velocity was 3 μm/s, and the cell–substrate contact time was 0.5 s. A representative result of 10 independent experiments is depicted.

Mentions: To examine the effects of disrupting the α4–paxillin interaction at a single molecule level and in the presence of an external force other than shear stress, we next measured the force of unitary adhesive interactions between wt α4β1 or the α4(Y991A)β1 mutant and immobilized VCAM-1 by atomic force microscopy (AFM). JB4-wt or JB4-α(Y991A) cells were coupled to the end of an AFM cantilever (Fig. 8 A) and lowered onto a VCAM-1–Fc-coated surface. After a 0.5-s contact, the frequency of productive adhesive events and their strength were analyzed by the degree of deflection experienced by the cantilever during its retraction from the adhesive substrate. Both cell types detached from the VCAM-1 substrate through single jumps, suggesting the breakage of individual bonds during cantilever retraction (Fig. 8 B). The adhesion frequencies of all experiments were maintained below 30%, a level assumed to reflect single α4β1–VCAM-1 interactions (Zhang et al., 2004). The specificity of the adhesive events detected in this system were confirmed by a similar 70% reduction in total binding events by the blockade of wt α4β1 with Bio1211 (Lin et al., 1999) or by omission of VCAM-1 from the substrate (Fig. 8 C and not depicted). As indicated by the force histograms derived for JB4-wt or JB4-α4(Y991A) cells (Fig. 8 C), the frequency of productive adhesive events developed by α4(Y991A)β1 was up to 10-fold lower than those developed by α4β1 after background subtraction. The distribution of unbinding (rupture) forces measured for the two integrin variants was, however, similar (Fig. 8 C). Thus, paxillin association with the α4 subunit dramatically augments the ability of α4β1 to form adhesive tethers that resist disruptive forces irrespective to whether these forces are applied during a vertical force loading (AFM) or during cell rotation (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)

α4(Y991A)β1 fails to stabilize bonds ruptured by an AFM probe. (A) Schematic representation of the experimental system. JB4 cells were coupled to an AFM cantilever tip via an anti-CD43 mAb. VCAM-Fc was immobilized onto the substrate as in previous figures. (B) Representative AFM force–displacement curves acquired with wt α4–expressing JB4 cells (top) or α4(Y991A)-expressing cells (middle) approaching the VCAM-1–Fc-bearing substrate. A force–displacement curve of wt α4–expressing JB4 approaching a control substrate devoid of VCAM-1 is indicated in the bottom curve. (C) Force histograms of α4β1–VCAM-1 unbinding forces measured under a fixed loading rate of 0.33 nN/s. The number of productive adhesive interactions and their unbinding force distribution are depicted. Background binding is depicted by the dashed line. The mean unbinding force (UF) values of 10 independent experiments are indicated near each histogram. Pulling velocity was 3 μm/s, and the cell–substrate contact time was 0.5 s. A representative result of 10 independent experiments is depicted.
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Related In: Results  -  Collection

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fig8: α4(Y991A)β1 fails to stabilize bonds ruptured by an AFM probe. (A) Schematic representation of the experimental system. JB4 cells were coupled to an AFM cantilever tip via an anti-CD43 mAb. VCAM-Fc was immobilized onto the substrate as in previous figures. (B) Representative AFM force–displacement curves acquired with wt α4–expressing JB4 cells (top) or α4(Y991A)-expressing cells (middle) approaching the VCAM-1–Fc-bearing substrate. A force–displacement curve of wt α4–expressing JB4 approaching a control substrate devoid of VCAM-1 is indicated in the bottom curve. (C) Force histograms of α4β1–VCAM-1 unbinding forces measured under a fixed loading rate of 0.33 nN/s. The number of productive adhesive interactions and their unbinding force distribution are depicted. Background binding is depicted by the dashed line. The mean unbinding force (UF) values of 10 independent experiments are indicated near each histogram. Pulling velocity was 3 μm/s, and the cell–substrate contact time was 0.5 s. A representative result of 10 independent experiments is depicted.
Mentions: To examine the effects of disrupting the α4–paxillin interaction at a single molecule level and in the presence of an external force other than shear stress, we next measured the force of unitary adhesive interactions between wt α4β1 or the α4(Y991A)β1 mutant and immobilized VCAM-1 by atomic force microscopy (AFM). JB4-wt or JB4-α(Y991A) cells were coupled to the end of an AFM cantilever (Fig. 8 A) and lowered onto a VCAM-1–Fc-coated surface. After a 0.5-s contact, the frequency of productive adhesive events and their strength were analyzed by the degree of deflection experienced by the cantilever during its retraction from the adhesive substrate. Both cell types detached from the VCAM-1 substrate through single jumps, suggesting the breakage of individual bonds during cantilever retraction (Fig. 8 B). The adhesion frequencies of all experiments were maintained below 30%, a level assumed to reflect single α4β1–VCAM-1 interactions (Zhang et al., 2004). The specificity of the adhesive events detected in this system were confirmed by a similar 70% reduction in total binding events by the blockade of wt α4β1 with Bio1211 (Lin et al., 1999) or by omission of VCAM-1 from the substrate (Fig. 8 C and not depicted). As indicated by the force histograms derived for JB4-wt or JB4-α4(Y991A) cells (Fig. 8 C), the frequency of productive adhesive events developed by α4(Y991A)β1 was up to 10-fold lower than those developed by α4β1 after background subtraction. The distribution of unbinding (rupture) forces measured for the two integrin variants was, however, similar (Fig. 8 C). Thus, paxillin association with the α4 subunit dramatically augments the ability of α4β1 to form adhesive tethers that resist disruptive forces irrespective to whether these forces are applied during a vertical force loading (AFM) or during cell rotation (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