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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 poorly associates with talin. (A) The α4(Y991A)β1 complex does not properly recruit talin. Total lysates (left) or talin coprecipitating with anti-α4, anti-β1, or an irrelevant mouse IgG (right) from lysates of either JB4 transfected with wt α4 or the α4(Y991A) mutant (top). The blot was stripped and reprobed for α4 (bottom). (B) Silencing of talin in JB4 cells expressing either wt or α4(Y991A). The indicated cells were transfected with either talin1-specific or control siRNA. Total lysates of each group were immunoblotted with talin or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 66 and 67% in talin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Talin suppression preferentially impairs wt α4β1–mediated resistance to detachment from sVCAM-1 (2,220 sites/μm2). *, P < 0.03 for control compared with talin-silenced cells at 0.5 dyn/cm2. Where indicated, cells were pretreated with the α4β1-specific blocker BIO1211. (inset) Effect of talin suppression on resistance to detachment from VCAM-1–Fc (30 CAM sites/μm2) of JB4 cells expressing either wt or α4(Y991A). In each panel, the mean ± range of two experimental fields is depicted. Results are representative of three independent experiments. Error bars represent SD.
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fig5: The α4(Y991A)β1 mutant poorly associates with talin. (A) The α4(Y991A)β1 complex does not properly recruit talin. Total lysates (left) or talin coprecipitating with anti-α4, anti-β1, or an irrelevant mouse IgG (right) from lysates of either JB4 transfected with wt α4 or the α4(Y991A) mutant (top). The blot was stripped and reprobed for α4 (bottom). (B) Silencing of talin in JB4 cells expressing either wt or α4(Y991A). The indicated cells were transfected with either talin1-specific or control siRNA. Total lysates of each group were immunoblotted with talin or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 66 and 67% in talin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Talin suppression preferentially impairs wt α4β1–mediated resistance to detachment from sVCAM-1 (2,220 sites/μm2). *, P < 0.03 for control compared with talin-silenced cells at 0.5 dyn/cm2. Where indicated, cells were pretreated with the α4β1-specific blocker BIO1211. (inset) Effect of talin suppression on resistance to detachment from VCAM-1–Fc (30 CAM sites/μm2) of JB4 cells expressing either wt or α4(Y991A). In each panel, the mean ± range of two experimental fields is depicted. Results are representative of three independent experiments. Error bars represent SD.

Mentions: In light of this poor cytoskeletal anchorage of α4(Y991A)β1, we next compared the level of talin associated with the wt or mutant α4β1 complex in nonadherent Jurkat cells. Notably, constitutive talin binding to the α4(Y991A)β1 complex was significantly reduced compared with the wt integrin, as was evident from coprecipitation analysis (Fig. 5 A). Knocking down up to 65% of the total talin content in wt α4β1–expressing Jurkat cells (Fig. 5 B) retained integrin expression (not depicted) but resulted in significant reduction in their α4β1-mediated shear resistance on both sVCAM-1 and VCAM-1–Fc (Fig. 5 C, left and right insets, respectively). Notably, identical suppression of talin expression in the α4(Y991A)β1-expressing Jurkat cells (Fig. 5 B) had no effect on their low shear resistant adhesion to identical VCAM-1 substrates (Fig. 5 C, right). These results collectively suggest that paxillin association with α4β1 also recruits talin to the α4–paxillin complex and may enhance talin association with the β1 subunit tail. Thus, both paxillin and talin associations promote α4β1-dependent cell resistance to detachment from 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 poorly associates with talin. (A) The α4(Y991A)β1 complex does not properly recruit talin. Total lysates (left) or talin coprecipitating with anti-α4, anti-β1, or an irrelevant mouse IgG (right) from lysates of either JB4 transfected with wt α4 or the α4(Y991A) mutant (top). The blot was stripped and reprobed for α4 (bottom). (B) Silencing of talin in JB4 cells expressing either wt or α4(Y991A). The indicated cells were transfected with either talin1-specific or control siRNA. Total lysates of each group were immunoblotted with talin or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 66 and 67% in talin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Talin suppression preferentially impairs wt α4β1–mediated resistance to detachment from sVCAM-1 (2,220 sites/μm2). *, P < 0.03 for control compared with talin-silenced cells at 0.5 dyn/cm2. Where indicated, cells were pretreated with the α4β1-specific blocker BIO1211. (inset) Effect of talin suppression on resistance to detachment from VCAM-1–Fc (30 CAM sites/μm2) of JB4 cells expressing either wt or α4(Y991A). In each panel, the mean ± range of two experimental fields is depicted. Results are representative of three independent experiments. Error bars represent SD.
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fig5: The α4(Y991A)β1 mutant poorly associates with talin. (A) The α4(Y991A)β1 complex does not properly recruit talin. Total lysates (left) or talin coprecipitating with anti-α4, anti-β1, or an irrelevant mouse IgG (right) from lysates of either JB4 transfected with wt α4 or the α4(Y991A) mutant (top). The blot was stripped and reprobed for α4 (bottom). (B) Silencing of talin in JB4 cells expressing either wt or α4(Y991A). The indicated cells were transfected with either talin1-specific or control siRNA. Total lysates of each group were immunoblotted with talin or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 66 and 67% in talin content in JB4 expressing either wt or α4(Y991A), respectively. (C) Talin suppression preferentially impairs wt α4β1–mediated resistance to detachment from sVCAM-1 (2,220 sites/μm2). *, P < 0.03 for control compared with talin-silenced cells at 0.5 dyn/cm2. Where indicated, cells were pretreated with the α4β1-specific blocker BIO1211. (inset) Effect of talin suppression on resistance to detachment from VCAM-1–Fc (30 CAM sites/μm2) of JB4 cells expressing either wt or α4(Y991A). In each panel, the mean ± range of two experimental fields is depicted. Results are representative of three independent experiments. Error bars represent SD.
Mentions: In light of this poor cytoskeletal anchorage of α4(Y991A)β1, we next compared the level of talin associated with the wt or mutant α4β1 complex in nonadherent Jurkat cells. Notably, constitutive talin binding to the α4(Y991A)β1 complex was significantly reduced compared with the wt integrin, as was evident from coprecipitation analysis (Fig. 5 A). Knocking down up to 65% of the total talin content in wt α4β1–expressing Jurkat cells (Fig. 5 B) retained integrin expression (not depicted) but resulted in significant reduction in their α4β1-mediated shear resistance on both sVCAM-1 and VCAM-1–Fc (Fig. 5 C, left and right insets, respectively). Notably, identical suppression of talin expression in the α4(Y991A)β1-expressing Jurkat cells (Fig. 5 B) had no effect on their low shear resistant adhesion to identical VCAM-1 substrates (Fig. 5 C, right). These results collectively suggest that paxillin association with α4β1 also recruits talin to the α4–paxillin complex and may enhance talin association with the β1 subunit tail. Thus, both paxillin and talin associations promote α4β1-dependent cell resistance to detachment from 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