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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 mediates poor shear-resistant adhesion to VCAM-1. (A) JB4 Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) were settled for 1 min on low density VCAM-1–Fc (80 CAM sites/μm2; left) or on sVCAM-1 coated at medium (1,480 sites/μm2; middle) or high density (3,700 sites/μm2; right), and their resistance to detachment by incremented shear stresses was analyzed. The fraction of cells within initially settled populations remaining bound at the end of each interval of shear increase is shown for each cell population. (B) FACS staining of ectopically expressed α4, endogenous β1 and αL subunits, as well as of the β1 activation neoepitope 15/7 on wt- and α4(Y991A)-expressing JB4 cells, depicted with black and gray lines, respectively. (C) LFA-1–dependent adhesion of both wt- and α4(Y991A)-expressing JB4 cells to low (80 sites/μm2) or medium density ICAM-1–Fc (160 sites/μm2) as well as to high density ICAM-1 (7,600 sites/μm2), measured as in A. In each panel, the mean ± range of two experimental fields is depicted. Results in A and C are representative of six independent experiments. (D) FACS staining of VCAM-1, ICAM-1, and E-selectin on TNFα-stimulated HUVECs. Dotted lines represent staining of isotype-matched controls (left). VLA-4–dependent adhesion of JB4 cells transfected with wt α4 (WT) or the α4(Y991A) mutant to intact (left) or E-selectin–blocked TNFα-stimulated HUVECs (right). Resistance to the detachment of cells settled for 1 min on the monolayer was assessed as in A. Shown in parenthesis are the fractions of adherent cells that maintained rolling on the different HUVECs at 5 dyn/cm2. LFA-1 blockage did not affect Jurkat resistance to detachment, whereas pretreatment with the α4β1-specific blocker Bio1211 (at 1 μg/ml) resulted in complete loss of shear resistance (not depicted). Error bars represent SD.
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fig1: The α4(Y991A)β1 mutant mediates poor shear-resistant adhesion to VCAM-1. (A) JB4 Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) were settled for 1 min on low density VCAM-1–Fc (80 CAM sites/μm2; left) or on sVCAM-1 coated at medium (1,480 sites/μm2; middle) or high density (3,700 sites/μm2; right), and their resistance to detachment by incremented shear stresses was analyzed. The fraction of cells within initially settled populations remaining bound at the end of each interval of shear increase is shown for each cell population. (B) FACS staining of ectopically expressed α4, endogenous β1 and αL subunits, as well as of the β1 activation neoepitope 15/7 on wt- and α4(Y991A)-expressing JB4 cells, depicted with black and gray lines, respectively. (C) LFA-1–dependent adhesion of both wt- and α4(Y991A)-expressing JB4 cells to low (80 sites/μm2) or medium density ICAM-1–Fc (160 sites/μm2) as well as to high density ICAM-1 (7,600 sites/μm2), measured as in A. In each panel, the mean ± range of two experimental fields is depicted. Results in A and C are representative of six independent experiments. (D) FACS staining of VCAM-1, ICAM-1, and E-selectin on TNFα-stimulated HUVECs. Dotted lines represent staining of isotype-matched controls (left). VLA-4–dependent adhesion of JB4 cells transfected with wt α4 (WT) or the α4(Y991A) mutant to intact (left) or E-selectin–blocked TNFα-stimulated HUVECs (right). Resistance to the detachment of cells settled for 1 min on the monolayer was assessed as in A. Shown in parenthesis are the fractions of adherent cells that maintained rolling on the different HUVECs at 5 dyn/cm2. LFA-1 blockage did not affect Jurkat resistance to detachment, whereas pretreatment with the α4β1-specific blocker Bio1211 (at 1 μg/ml) resulted in complete loss of shear resistance (not depicted). Error bars represent SD.

Mentions: Paxillin binding to the α4-cytoplasmic domain is important for integrin α4β1 signaling but not for adhesion developed in shear-free conditions (Rose et al., 2003). To examine the role of paxillin binding in α4β1-mediated adhesion under shear stress, we analyzed the resistance to shear-induced detachment from the α4β1 ligand VCAM-1 of α4-deficient JB4 Jurkat T cells transfected with either wild-type (wt) α4 (JB4-wt) or the paxillin binding–defective α4(Y991A) mutant JB4-α4(Y991A) (Rose et al., 2003). JB4-α4(Y991A) cells were less resistant to shear-induced detachment than their JB4-wt counterparts (Fig. 1 A). Notably, bivalent VCAM-1 (VCAM-1–Fc) was much more potent than monovalent soluble VCAM-1 (sVCAM-1) in supporting α4β1-specific adhesion (Fig. 1 A), but it still could not rescue the adhesive defect of the α4(Y991A) mutant. These results were confirmed with multiple clones expressing similar levels of α4 and β1 subunits as well as the β1 activation epitope 15/7 (Fig. 1 B and not depicted). Nevertheless, resistance to detachment from different densities of either ICAM-1–Fc or ICAM-1 was comparable between wt- and mutant α4β1–expressing cells (Fig. 1 C). In agreement with these results, VLA-4–dependent adhesion to TNFα-stimulated human umbilical vein endothelial cells (HUVECs) was reduced in Jurkat cells expressing the α4(Y991A) mutant (Fig. 1 D), in particular at shear stresses ≥5 dyn/cm2, within the upper range of shear stresses prevailing in postcapillary venules where the majority of lymphocyte extravasation takes place (Firrell and Lipowsky, 1989). Whereas most cells expressing the wt α4 firmly arrested on the stimulated HUVEC via their VLA-4, a significant fraction of α4(Y991A) mutant–expressing cells failed to arrest and established endothelial (E)-selectin–dependent rolling on the HUVEC (Fig. 1 D). In the absence of functional E-selectin, the shear resistance of cells expressing the α4(Y991A) mutant was much lower than the shear resistance of cells expressing wt α4 (Fig. 1 D). Because the contribution of LFA-1 to Jurkat arrest was minimal, these data suggest that the Y991A α4 mutant is deficient in establishing α4β1-mediated shear resistance on endothelial cells expressing VCAM-1 as well as on substrates coated with isolated 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 mediates poor shear-resistant adhesion to VCAM-1. (A) JB4 Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) were settled for 1 min on low density VCAM-1–Fc (80 CAM sites/μm2; left) or on sVCAM-1 coated at medium (1,480 sites/μm2; middle) or high density (3,700 sites/μm2; right), and their resistance to detachment by incremented shear stresses was analyzed. The fraction of cells within initially settled populations remaining bound at the end of each interval of shear increase is shown for each cell population. (B) FACS staining of ectopically expressed α4, endogenous β1 and αL subunits, as well as of the β1 activation neoepitope 15/7 on wt- and α4(Y991A)-expressing JB4 cells, depicted with black and gray lines, respectively. (C) LFA-1–dependent adhesion of both wt- and α4(Y991A)-expressing JB4 cells to low (80 sites/μm2) or medium density ICAM-1–Fc (160 sites/μm2) as well as to high density ICAM-1 (7,600 sites/μm2), measured as in A. In each panel, the mean ± range of two experimental fields is depicted. Results in A and C are representative of six independent experiments. (D) FACS staining of VCAM-1, ICAM-1, and E-selectin on TNFα-stimulated HUVECs. Dotted lines represent staining of isotype-matched controls (left). VLA-4–dependent adhesion of JB4 cells transfected with wt α4 (WT) or the α4(Y991A) mutant to intact (left) or E-selectin–blocked TNFα-stimulated HUVECs (right). Resistance to the detachment of cells settled for 1 min on the monolayer was assessed as in A. Shown in parenthesis are the fractions of adherent cells that maintained rolling on the different HUVECs at 5 dyn/cm2. LFA-1 blockage did not affect Jurkat resistance to detachment, whereas pretreatment with the α4β1-specific blocker Bio1211 (at 1 μg/ml) resulted in complete loss of shear resistance (not depicted). Error bars represent SD.
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Related In: Results  -  Collection

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fig1: The α4(Y991A)β1 mutant mediates poor shear-resistant adhesion to VCAM-1. (A) JB4 Jurkat cells expressing either wt α4 (WT) or the α4(Y991A) mutant (Y991A) were settled for 1 min on low density VCAM-1–Fc (80 CAM sites/μm2; left) or on sVCAM-1 coated at medium (1,480 sites/μm2; middle) or high density (3,700 sites/μm2; right), and their resistance to detachment by incremented shear stresses was analyzed. The fraction of cells within initially settled populations remaining bound at the end of each interval of shear increase is shown for each cell population. (B) FACS staining of ectopically expressed α4, endogenous β1 and αL subunits, as well as of the β1 activation neoepitope 15/7 on wt- and α4(Y991A)-expressing JB4 cells, depicted with black and gray lines, respectively. (C) LFA-1–dependent adhesion of both wt- and α4(Y991A)-expressing JB4 cells to low (80 sites/μm2) or medium density ICAM-1–Fc (160 sites/μm2) as well as to high density ICAM-1 (7,600 sites/μm2), measured as in A. In each panel, the mean ± range of two experimental fields is depicted. Results in A and C are representative of six independent experiments. (D) FACS staining of VCAM-1, ICAM-1, and E-selectin on TNFα-stimulated HUVECs. Dotted lines represent staining of isotype-matched controls (left). VLA-4–dependent adhesion of JB4 cells transfected with wt α4 (WT) or the α4(Y991A) mutant to intact (left) or E-selectin–blocked TNFα-stimulated HUVECs (right). Resistance to the detachment of cells settled for 1 min on the monolayer was assessed as in A. Shown in parenthesis are the fractions of adherent cells that maintained rolling on the different HUVECs at 5 dyn/cm2. LFA-1 blockage did not affect Jurkat resistance to detachment, whereas pretreatment with the α4β1-specific blocker Bio1211 (at 1 μg/ml) resulted in complete loss of shear resistance (not depicted). Error bars represent SD.
Mentions: Paxillin binding to the α4-cytoplasmic domain is important for integrin α4β1 signaling but not for adhesion developed in shear-free conditions (Rose et al., 2003). To examine the role of paxillin binding in α4β1-mediated adhesion under shear stress, we analyzed the resistance to shear-induced detachment from the α4β1 ligand VCAM-1 of α4-deficient JB4 Jurkat T cells transfected with either wild-type (wt) α4 (JB4-wt) or the paxillin binding–defective α4(Y991A) mutant JB4-α4(Y991A) (Rose et al., 2003). JB4-α4(Y991A) cells were less resistant to shear-induced detachment than their JB4-wt counterparts (Fig. 1 A). Notably, bivalent VCAM-1 (VCAM-1–Fc) was much more potent than monovalent soluble VCAM-1 (sVCAM-1) in supporting α4β1-specific adhesion (Fig. 1 A), but it still could not rescue the adhesive defect of the α4(Y991A) mutant. These results were confirmed with multiple clones expressing similar levels of α4 and β1 subunits as well as the β1 activation epitope 15/7 (Fig. 1 B and not depicted). Nevertheless, resistance to detachment from different densities of either ICAM-1–Fc or ICAM-1 was comparable between wt- and mutant α4β1–expressing cells (Fig. 1 C). In agreement with these results, VLA-4–dependent adhesion to TNFα-stimulated human umbilical vein endothelial cells (HUVECs) was reduced in Jurkat cells expressing the α4(Y991A) mutant (Fig. 1 D), in particular at shear stresses ≥5 dyn/cm2, within the upper range of shear stresses prevailing in postcapillary venules where the majority of lymphocyte extravasation takes place (Firrell and Lipowsky, 1989). Whereas most cells expressing the wt α4 firmly arrested on the stimulated HUVEC via their VLA-4, a significant fraction of α4(Y991A) mutant–expressing cells failed to arrest and established endothelial (E)-selectin–dependent rolling on the HUVEC (Fig. 1 D). In the absence of functional E-selectin, the shear resistance of cells expressing the α4(Y991A) mutant was much lower than the shear resistance of cells expressing wt α4 (Fig. 1 D). Because the contribution of LFA-1 to Jurkat arrest was minimal, these data suggest that the Y991A α4 mutant is deficient in establishing α4β1-mediated shear resistance on endothelial cells expressing VCAM-1 as well as on substrates coated with isolated 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