Talin 1 and Paxillin Facilitate Distinct Steps in Rapid VLA-4-mediated Adhesion Strengthening to Vascular Cell Adhesion Molecule 1

Abstract

VLA-4 (␣ 4 ␤ 1 ) is a key integrin in lymphocytes, interacting with endothelial vascular cell adhesion molecule 1 (VCAM-1) on blood vessels and stroma. To dissect the contribution of the two cytoskeletal VLA-4 adaptor partners paxillin and talin to VLA-4 adhesiveness, we transiently knocked them down in Jurkat T cells and primary resting human T cells by small interfering RNA silencing. Paxillin was required for VLA-4 adhesiveness to low density VCAM-1 under shear stress conditions and was found to control mechanical stability of bonds mediated by the ␣ 4 subunit but did not affect the integrin affinity or avidity to VCAM-1 in shear-free conditions. Talin 1 maintained VLA-4 in a high affinity conformation, thereby promoting rapid VLA-4 adhesion strengthening to VCAM-1 under both shear stress and shear-free conditions. Talin 1, but not paxillin, was required for VLA-4 to undergo optimal stimulation by the prototypic chemokine, CXCL12, under shear stress conditions. Interestingly, talin 1 and paxillin played the same distinct roles in VLA-4 adhesions of primary T lymphocytes, although VLA-4 affinity to VCAM-1 was at least 200-fold lower in these cells than in Jurkat cells. Collectively, our results suggest that whereas paxillin is a mechanical regulator of VLA-4 bonds generated in the absence of chemokine signals and low VCAM-1 occupancy, talin 1 is a versatile VLA-4 affinity regulator implicated in both spontaneous and chemokine-triggered rapid adhesions to VCAM-1.

Talin1 is required for integrin-dependent B lymphocyte homing to lymph nodes and the bone marrow but not for follicular B-cell maturation in the spleen

“…B-cell talin1 is also dispensable for the residual VLA-4 adhesiveness to VCAM-1 retained by nonstimulated B cells but is still critical for residual LFA-1 adhesiveness. In human T cells, this spontaneous VLA-4 adhesiveness is reduced by transient suppression of either talin1 or paxillin, 40 a specific adaptor of the ␣ 4 subunit of VLA-4 41 critical for its spontaneous adhesiveness, particularly under shear forces. 40 It is therefore possible that, in our conditional talin1 depletion B-cell model, prolonged depletion of talin1 allows paxillin to substitute for talin1 in conferring low spontaneous adhesiveness of VLA-4 but not of LFA-1.…”

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions

“…[5][6][7]9,15 Indeed, both chemokine-triggered and ligand-mediated activation of LFA-1, shown in this study to be defective in LAD-III Kindlin-3-deficient T cells, are impaired in T cells lacking talin1. 27 Similarly, VLA-4 activation by chemokines, which is also highly sensitive to talin1 depletion, 33 is also impaired in Kindlin-3-deficient LAD T cells. Nevertheless, VLA-4 is far less susceptible than LFA-1 to loss of Kindlin-3, as considerable VLA-4 activation, but not of LFA-1 activation, is retained in Kindlin-3-null effector T cells (supplemental Figure 6).…”

“…33 Talin1 binds VLA-4 at the ␤ subunit tail near the membrane-proximal NPXY motif, NPIY, whereas the putative Kindlin-3 binding site on this subunit is located close to the membrane distal NPKY site 8 ( Figure 5A). Based on the retained VLA-4-mediated tethering and rolling on VCAM-1 displayed by both Kindlin-3-null T and B lymphoblasts, we hypothesized that deletion of this membrane distal NPKY site would not affect VLA-4 binding to VCAM-1, whereas deletion of the talin1 binding site was likely to abrogate binding.…”

Talin at a glance

“…In T cells, talin 1 is required for T-cell receptor (TCR)-mediated regulation of the affinity, clustering and polarisation of integrin LFA-1 (a L β2), and small interfering RNA (siRNA)-mediated talin 1 knockdown impaired TCR-induced adhesion to and migration on ICAM1, and the conjugation of T cells to antigen-presenting cells (Smith et al, 2005;Simonson et al, 2006). Talin (but not vinculin) is also required for both F-actin and integrin accumulation at the immunological synapse (Nolz et al, 2007), and for the chemokine-induced increase in affinity of integrin VLA-4 (α4β1) for VCAM1, which is important in lymphocyte-endothelial cell adhesion (Manevich et al, 2007). In macrophages, talin 1 is required for phagocytosis that is mediated by αMβ2 integrin (complement receptor 3) (Lim et al, 2007), whereas a talin-PIPK1γ90 complex has been shown to play a novel role in clathrin-mediated endocytosis in the neuronal synapse (Morgan et al, 2004).…”