Intermediate-affinity LFA-1 binds α-actinin-1 to control migration at the leading edge of the T cell
2007; Springer Nature; Volume: 27; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7601959
ISSN1460-2075
AutoresPaula Stanley, Andrew Smith, Alison McDowall, Alastair Nicol, Daniel Zicha, Nancy Hogg,
Tópico(s)Immunotherapy and Immune Responses
ResumoArticle13 December 2007Open Access Intermediate-affinity LFA-1 binds α-actinin-1 to control migration at the leading edge of the T cell Paula Stanley Paula Stanley Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Andrew Smith Andrew Smith Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UKPresent address: Department of Medicine, University College London, London WC1E 6JJ, UK Search for more papers by this author Alison McDowall Alison McDowall Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Alastair Nicol Alastair Nicol Light Microscopy Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Daniel Zicha Daniel Zicha Light Microscopy Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Nancy Hogg Corresponding Author Nancy Hogg Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Paula Stanley Paula Stanley Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Andrew Smith Andrew Smith Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UKPresent address: Department of Medicine, University College London, London WC1E 6JJ, UK Search for more papers by this author Alison McDowall Alison McDowall Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Alastair Nicol Alastair Nicol Light Microscopy Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Daniel Zicha Daniel Zicha Light Microscopy Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Nancy Hogg Corresponding Author Nancy Hogg Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Author Information Paula Stanley1,‡, Andrew Smith1,‡, Alison McDowall1, Alastair Nicol2, Daniel Zicha2 and Nancy Hogg 1 1Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London, UK 2Light Microscopy Laboratory, Cancer Research UK London Research Institute, London, UK ‡These authors contributed equally to this work *Corresponding author. Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Tel.: +44 207 269 3255; Fax: +44 207 269 3093; E-mail: [email protected] The EMBO Journal (2008)27:62-75https://doi.org/10.1038/sj.emboj.7601959 Present address: Department of Medicine, University College London, London WC1E 6JJ, UK PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info T lymphocytes use LFA-1 to migrate into lymph nodes and inflammatory sites. To investigate the mechanisms regulating this migration, we utilize mAbs selective for conformational epitopes as probes for active LFA-1. Expression of the KIM127 epitope, but not the 24 epitope, defines the extended conformation of LFA-1, which has intermediate affinity for ligand ICAM-1. A key finding is that KIM127-positive LFA-1 forms new adhesions at the T lymphocyte leading edge. This LFA-1 links to the cytoskeleton through α-actinin-1 and disruption at the level of integrin or actin results in loss of cell spreading and migratory speed due to a failure of attachment at the leading edge. The KIM127 pattern contrasts with high-affinity LFA-1 that expresses both 24 and KIM127 epitopes, is restricted to the mid-cell focal zone and controls ICAM-1 attachment. Identification of distinctive roles for intermediate- and high-affinity LFA-1 in T lymphocyte migration provides a biological function for two active conformations of this integrin for the first time. Introduction T lymphocytes are continually recruited from the circulation into lymph nodes and other tissues in response to stimuli released at sites of injury or infection (von Andrian and Mempel, 2003; Cyster, 2005). This recruitment involves the integrin LFA-1 (CD11a/CD18; αLβ2) acting as a migratory receptor (Hogg et al, 2003; Dustin et al, 2004). If turnover of active to inactive LFA-1 is prevented, then motility decreases both in vivo and in vitro, indicating that conversion between different activity states is essential for lymphocyte migration (Semmrich et al, 2005; Smith et al, 2005). T lymphocytes move rapidly across the vasculature into lymph nodes where they can reach speeds of ∼40 μm/min (Mempel et al, 2004). This ability to migrate is vital for the ultimate success of the immune response as it increases the opportunity for cellular interactions, thereby improving the chance of antigen recognition. Integrin activation involves an alteration in conformation giving rise to an affinity increase, and adhesion strengthening through receptor clustering (Carman and Springer, 2003; Dustin et al, 2004; Kinashi, 2005). Crystallization of the β3 integrin αvβ3 has revealed details of the conformational changes that integrins undergo following activation (Xiong et al, 2001). A basic model for β2 integrin LFA-1 activation has been developed from structural data, together with observations from electron microscopic studies of isolated integrins and from the use of mAbs that recognize epitopes expressed upon activation (Takagi et al, 2002; Luo et al, 2007). Basically, one conformation of LFA-1 is bent at the junction between the thigh and calf-1 domains of the αL subunit and between EGF-like domains 1 and 2 of the β2 subunit. Upon activation, LFA-1 extends, exposing an epitope recognized by mAb KIM127 that is located in the EGF-like domain 2 at the bend in the β2 subunit (Robinson et al, 1992; Beglova et al, 2002). The extended KIM127+ LFA-1 adopts both closed and open conformations corresponding to intermediate- and high-affinity forms of LFA-1, respectively (Kinashi, 2006; Luo et al, 2007). The epitope recognized by mAb 24 is located in a loop at the top of the I-like domain near the MIDAS site in the β2 subunit (Lu et al, 2001; Kamata et al, 2002), and its expression is characteristic of the high-affinity conformation of LFA-1 (Dransfield et al, 1992b; Lu et al, 2001; Hogg et al, 2002; Kamata et al, 2002). This epitope is postulated to form upon interaction of the α subunit I domain with the β2 subunit I-like domain via a pulling mechanism, giving rise to the open, high-affinity form of integrin (Alonso et al, 2002; Luo et al, 2007). In order to attach to integrin ligands and migrate, primary T lymphocytes require stimulus from a chemoattractant such as a chemokine. However, for preactivated T lymphoblasts, LFA-1-mediated binding to an ICAM-1-expressing surface is sufficient to cause cell polarization and migration without the need for added chemokines (Dustin et al, 1997; Smith et al, 2003). Previously we have shown that a 'focal zone' containing high-affinity 24+ LFA-1 is located in the mid-cell region of the polarized T lymphocyte and influences the speed of migration (Smith et al, 2005). To date, little is known of the characteristics of LFA-1 expressed at the leading edge of migrating T lymphocytes. To investigate the nature of these adhesions and compare them with LFA-1 in the focal zone, we have made use of two well-described mAbs, KIM127 and 24, as probes for intermediate- and high-affinity LFA-1. We find that they detect these two forms of active LFA-1 in distinct regions of the migrating cell. Results Expression of LFA-1 activation epitopes KIM127 and 24 on T cells A comparison was made of the expression patterns of LFA-1 activation epitopes recognized by mAbs KIM127 and 24 on T cells migrating on the LFA-1 ligand ICAM-1. Both freshly derived primary T lymphocytes and cultured T lymphoblasts, that are referred to in this study as T cells, were investigated. When analyzed by flow cytometry, the majority of T cells expressed both epitopes (60–95%; n=10), whereas a lower percentage of freshly derived T lymphocytes expressed the epitopes (10–40%; n=4). These percentages correlated with the proportions of cells able to attach to immobilized ICAM-1 (data not shown). Thus, the cells that attach and migrate represent pre-existing subsets on which LFA-1 displays activation epitopes. The next step was to compare the distribution of the LFA-1 activation epitopes on T cells that were allowed to migrate before fixation and subsequent immunostaining. MAb 24+ LFA-1 was restricted to the mid-cell focal zone both for unstimulated, cultured T cells as previously described (Smith et al, 2005) and primary T lymphocytes that had been stimulated with the chemokine SDF-1 (CXCL12) (Figure 1). In contrast, mAb KIM127 labelled LFA-1 throughout the lamellar region, as well as intensely labelling the focal zone (Figure 1). Therefore, the extended intermediate-affinity form of LFA-1 that expresses the KIM127, but not 24, epitope is localized to the spreading lamella of the T cell, whereas high-affinity LFA-1 that expresses both epitopes is restricted to the focal zone. The distribution patterns of LFA-1 epitopes show that these two conformations of active LFA-1 have distinct locations on the migrating T cell. Figure 1.Expression of LFA-1 activation epitopes on migrating T lymphocytes. T cells and freshly isolated primary T lymphocytes migrating on ICAM-1Fc-coated coverslips were fixed and permeabilized before labelling with either KIM127–Alexa546 or 24–Alexa546 addition. Freshly isolated T lymphocytes were exposed for 5 min to SDF-1 (CXCL12) to induce a migratory phenotype. Images are shown in a rainbow false color scale, with the highest expression in red and the lowest blue. The enlarged area of each leading edge is shown with a white box. Scale bar=10 μm. Download figure Download PowerPoint As well as acting as activation reporters, both mAb 24 and KIM127 can stabilize the active forms of LFA-1 that they recognize (Dransfield et al, 1992a; Robinson et al, 1992; Smith et al, 2005; Nishida et al, 2006). When T cells were exposed to mAb KIM127 during migration, KIM127+ LFA-1 attachments (red) to ICAM-1 were formed at the leading edge (see asterisks). As the T cell lamellae extended and made further attachments, KIM127+ staining increased and the T cell rapidly stopped translocating forward (Figure 2A; Supplementary Figure 1 and Video 1). When the KIM127+ adhesions were stabilized to a certain level around one leading edge, another lamellipodial protrusion appeared that was in turn stabilized by making new KIM127+ attachments. Eventually when the leading edge membranes could no longer move in any direction, the cell reversed its polarity and the process was repeated until the T cell was unable to make any new KIM 127+ attachments. Figure 2.Effect of mAbs KIM127 and 24 on migrating T cells. (A) Live-cell imaging of T cells migrating on ICAM-1Fc-coated coverslips following addition of directly labelled mAb KIM127 at 0 s. Confocal images showing KIM127–Alexa546 (red) localization are overlaid with the phase images. The leading lamellipodia are indicated by *. Scale bar=10 μm. (B) Images as in panel A but following addition of 24–Alexa488 (green) at 0 s. (C) Addition of KIM127–Alexa488 (green) for 5 min before counterstaining with KIM127–Alexa546 (red) during fixation. Scale bar=5 μm. Download figure Download PowerPoint By comparison, when T cells were exposed to mAb 24 during migration, 24+ LFA-1 (green) was focused not at the leading edge but in the focal zone (Figure 2B; Supplementary Figure 1). Over time the focal zone extended forward as the number of new 24+ attachments increased. Of note is the observation that KIM127 does not have access to its epitope in the focal zone area as might be expected. However, if the migrating cell is subsequently fixed and then stained for KIM127, the epitope is detected in the focal zone in keeping with the pattern of immunostaining shown in Figure 1 (data not shown). There may be a lack of access of KIM127 in the focal zone region because of the positioning of the epitope on LFA-1 or because packing of LFA-1 molecules in this region interferes with mAb binding. To investigate the formation of KIM127+ adhesions in more detail, we carried out a two-stage experiment focusing on the interface with ICAM-1. T cells were allowed to migrate in an excess amount of KIM127-Alexa488 (green) to label the available adhesions. Unbound mAb was then washed out, and, to capture the adhesions formed subsequently, KIM127-Alexa546 (red) was added while the cells were being fixed. Under these conditions, KIM127+ adhesions (red) were only observed at the leading edge confirming that new KIM127+ adhesions form in this location and are stabilized by the mAb (Figure 2C). This stabilizing effect then causes the unattached leading edge of the T cell to move in another direction over pre-existing stabilized LFA-1 adhesions to form new ones. We next used interference reflection microscopy (IRM) to focus on the interface between LFA-1 on migrating T cells and ICAM-1 at the leading edge following addition of KIM127. At 0 s the T cell migrates in one direction, with its leading edge indicated by an arrow (Figure 3; Supplementary Video 2). After 30 s of exposure to KIM127, LFA-1 at the leading edge has become stabilized. The LFA-1 attachment points are seen as dark contrast regions and a new leading edge moves the cell in another direction. At 70 s, the cell changes direction again as the second leading edge becomes stabilized. Finally at 130 s the LFA-1 adhesions are stabilized all around the cell and it has come to a halt. We conclude that mAb KIM127 prevents turnover of LFA-1/ICAM-1 attachments through its ability to stabilize LFA-1 adhesions at the leading edge. Addition of mAb 24 has no effect on the leading edge (data not shown). Figure 3.Effect of mAb KIM127 on IRM imaging of a T cell migrating on ICAM-1. The image of a single representative T cell migrating on an ICAM-1-coated coverslip is shown with 10 μg/ml KIM127 added at 0 s. KIM127 stabilizes adhesions of leading edge 1 (see arrow) at 30 s shown by the formation of dark contact areas. The cell alters direction, forming leading edge 2, which also becomes stabilized (70 s). This is followed by stabilization of leading edge 3 (130 s) and the process continues until the cell can no longer move. Scale bar=5 μm. Download figure Download PowerPoint In contrast to both the 24+ and KIM127+ adhesions, the majority of which are restricted to LFA-1 in contact with ICAM-1, total LFA-1 was distributed over the entire cell surface (Supplementary Figure 2). The lamella at the leading edge displayed a lower level of LFA-1 compared with the focal zone and the uropod at the rear as previously reported (Smith et al, 2005). Therefore, for T cells migrating on ICAM-1, the presence of mAbs recognizing activation epitopes causes build up of LFA-1 adhesions at the leading edge for KIM127, and at the focal zone, for 24. A role for intermediate-affinity LFA-1 in random T cell migration and chemotaxis As mAbs 24 and KIM127 labelled distinct LFA-1 conformations in different locations when T cells were migrating on ICAM-1, we asked whether the way in which these mAbs affected migration could provide information about the differing roles of the two forms of LFA-1. Non-blocking anti-LFA-1 mAb YTH 81.5 had no effect, but KIM127 caused a 63±4% reduction in speed of migration (Figure 4A) by preventing the translocation of individual T cells (Figure 4B). In contrast, there was no significant alteration in cell speed after the addition of 24 for the initial 10-min period (Figure 4A and B), but longer exposure gradually resulted in reduced migration (data not shown). To recreate more closely a potential in vivo situation, the effect of these mAbs on T cell migration through ICAM-1-coated filters in response to chemokine SDF-1 was tested (Figure 4C). SDF-1-induced migration was inhibited 96.4±0.7% by KIM127, 45.7±1.0% by 24 and was unaffected by YTH 81.5 (Figure 4C). Figure 4.Effect of KIM127 and 24 mAbs on T cell migration and chemotaxis. (A) Speed of migration of T cells on ICAM-1Fc-coated coverslips in the first 10 min following addition of LFA-1 mAbs YTH81.5, KIM127 or 24. Migration speed was calculated for 10 cells per treatment and expressed as a percentage of control cells. Data from a representative experiment are shown (n=3; *P<0.01). (B) Migratory tracks of individual T cells in the presence of mAbs YTH81.5, KIM127 or 24 from the same experiment as in panel A. (C) T cell chemotaxis for 60 min across ICAM-1Fc-coated filters in response to 10 nM SDF-1 in the presence or absence of the indicated LFA-1 mAbs. Each condition was run in duplicate. A representative experiment (n=3) is shown. (D) Live-cell imaging of T cells migrating on TNF α-activated-HUVECs following addition of fluorescently labelled mAbs (0–180 s). Confocal images of KIM127 (red) or 24 (green) localization are overlaid with phase images. The leading edge is indicated by * and shedding of 24-positive LFA-1 from the trailing edge by white arrows. Scale bar=10 μm. Download figure Download PowerPoint Confocal microscopy was used to observe T cells migrating on TNF-α-activated human umbilical vein endothelial cells (HUVECs) following exposure to fluorescently labelled KIM127 or 24 (Figure 4D; Supplementary Videos 3A and B). The cells that bound KIM127 failed to translocate, showing that KIM127 effectively inhibited turnover of LFA-1 adhesions to the endothelial cell surface. In contrast, mAb 24 stabilized only the mid-cell focal zone LFA-1, leaving the leading edge free to make fresh attachments and move forward. In fact the strength of this motility was sufficient to overcome the 24-stabilized LFA-1 adhesions, a proportion of which were left behind on the endothelium (see arrows). These results further underline the importance of intermediate-affinity KIM127+ LFA-1 in making adhesions at the front of the cell and show that this LFA-1 controls the ability of T cells to migrate. KIM127+ LFA-1 at the T cell leading edge is linked to α-actinin-1 Given the dependence of LFA-1-mediated migration on reorganization of the cytoskeleton (Porter et al, 2002; Smith et al, 2003) and the dramatic effect of KIM127 on T cell migration, an issue was how intermediate-affinity LFA-1 might be linked into the cytoskeleton. Pavalko and co-workers have demonstrated binding of the neutrophil β2 integrin subunit to both talin and α-actinin (Sampath et al, 1998). As the focal zone LFA-1 is associated with talin (Smith et al, 2005), we explored whether α-actinin-1 might be associated with KIM127+ LFA-1. The T cell line HSB-2 was transfected with α-actinin-1–GFP (Guvakova et al, 2002) and the migrating cells exposed to mAb KIM127 while being fixed so that the mAb would label the newest adhesions. α-Actinin-1 was chiefly localized at the leading edge of the cell (green), overlapping with KIM127+ LFA-1 (red) (Figure 5A and B). Quantification of the expression levels confirmed that the highest levels of both molecules were at the leading edge (Figure 5B). When viewed as a vertical slice along the y-axis, it was apparent that colocalized KIM127+ LFA-1 and α-actinin-1 were not restricted to the interface with ICAM-1, but could also be found on non-attached membrane at the front of the cell (Figure 5A). As a control, GFP transfected into T cells was evenly distributed, indicating that the α-actinin–GFP pattern did not simply represent the volume occupied by ruffled membranes (data not shown). Figure 5.KIM127+LFA-1 binds to cytoskeletal protein α-actinin-1 at the leading edge of T cells. Confocal images of a representative HSB-2 cell transfected with α-actinin-1-GFP migrating on ICAM-1Fc and stained with KIM127–Alexa546 during fixation. (A) Z-stack and vertical slice along the y-axis of α-actinin–GFP (green) and KIM127+LFA-1-labelled (red) HSB-2 cells are shown. (B) Merged z-stack of α-actinin-GFP (green) and KIM127+LFA-1 (red) images. The level of expression of α-actinin-1 (green trace) and KIM127+ LFA-1 (red trace) along the length of the polarized HSB-2 T cell (indicated by red arrow on merged image) is recorded. Scale bar=10 μm. (C) Co-immununoprecipitation of αL and α-actinin-1. T cells were preincubated with primary mAb before lysis and western blotting. Top panel shows lysates of T cells preincubated with the following: lane 1, KIM127; lane 2, 24; lane 3, total LFA-1 mAb 38 and lane 4, control mAb 52 U. Western blotting using αL and α-actinin-1 mAbs shows the extent of co-immunoprecipitation. Middle and bottom panels show total αL and α-actinin-1 levels, respectively, from 10 μl of lysate (n=4). Download figure Download PowerPoint Further evidence for an association between LFA-1 and α-actinin-1 was sought by co-immunoprecipitation of T cell proteins using mAbs KIM127 and 24, pan-LFA-1 mAb 38 and an isotype control mAb (Figure 5C). α-Actinin-1 co-immunoprecipitated with KIM127+ and 38+ LFA-1, whereas the co-immunoprecipitation of α-actinin-1 with 24+ LFA-1 was at background levels (top panel). Loading control lanes for the LFA-1 αL subunit and α-actinin-1 are shown in the bottom panels. Using densitometry, we estimated that mAbs KIM127 and 38, but not mAb 24 or control mAb, immunoprecipitated 0.45±0.1% of total α-actinin-1 (n=3). The α-actinin-1 associated with LFA-1 therefore represents a small but distinctive subpopulation of this cytoskeleton-associated protein. Together, these results provide evidence that intermediate-affinity LFA-1 on T cells is associated with α-actinin-1 at the leading edge. T cell migration on ICAM-1 requires α-actinin-1 We asked whether α-actinin-1 was required for T cell migration and also in the step before migration that involves cell attachment from suspension onto immobilized ICAM-1. Small interfering RNA (siRNA) directed against α-actinin-1 was transfected into the HSB-2 T cell line, resulting in a 60% knockdown after 48 h (see arrow), but with no effect on talin expression (Figure 6A). The decreased level of α-actinin-1 did not impede T cell attachment and, in fact, caused a 35±5% increase in the level of adhesion (Figure 6B). However, when migration on ICAM-1 was assessed, α-actinin-1-knockdown T cells showed a 53±5% reduction in speed and a decreased ability of individual cells to translocate compared with control siRNA-transfected or untransfected cells (Figure 6C and D). These results are in contrast to knockdown of talin by 40% that produced a corresponding decrease in HSB-2 T cell attachment (Supplementary Figure 3), suggesting a key role for talin, and not α-actinin-1, in this step. Therefore α-actinin-1 has no role in the initial attachment to ICAM-1, but does have a major impact on the ability of the T cells to migrate. Figure 6.Effect of α-actinin-1 knockdown on T cell migration and adhesion on ICAM-1. (A) Western blot of total HSB-2 cell lysates from the following transfection steps: no siRNA, α-actinin-1 siRNA (ID 9416) or control siRNA transfectants probed for α-actinin-1, talin and α-tubulin. The level of α-actinin-1 knockdown was ∼60% (n=3). Two alternative siRNAs to α-actinin-1 (ID 147017 or ID 16804) knocked down by ∼30% (data not shown). (B) Adhesion to ICAM-1 of HSB-2 T cells transfected with no siRNA, control siRNA or α-actinin-1 siRNA (*P<0.01). The cells shown were from the same transfection as in panel A (representative experiment of n=3). (C) Average speed of HSB-2 cells transfected with no siRNA, control siRNA or α-actinin-1 siRNA (36 cells each; mean values of n=3; *P<0.01). (D) Cell tracks of migrating HSB-2 T cells transfected with control siRNA or siRNA specific for α-actinin-1 from the same data as in panel C and tracked for 10 min (12 cell tracks per condition). Download figure Download PowerPoint α-Actinin-1 links to the actin cytoskeleton during T cell migration To establish whether α-actinin-1 was linked to the actin cytoskeleton, we transfected HSB-2 T cells with α-actinin–GFP (green) and detected F-actin with phalloidin–Alexa546 (red). There was overlap at the leading edge between the highest cellular levels of α-actinin-1 and F-actin (Figure 7A). Next, T cells were transfected with an α-actinin-1 construct (α-actinin–GFP), a second α-actinin-1 construct lacking the actin-binding domain (ΔN-actinin–GFP) or GFP as a control. The T cells expressing ΔN-actinin–GFP showed a 75±9% decrease in speed compared with α-actinin–GFP and control GFP-expressing cells (Figure 7B). Tracking individual cells highlighted a loss of translocation associated with the ΔN-actinin–GFP compared with controls (Figure 7C). The cells expressing α-actinin–GFP or GFP had a polarized morphology, whereas the ΔN-α-actinin–GFP transfectants attached normally to ICAM-1, but remained rounded (Figure 7C). Therefore colocalization of α-actinin-1 and F-actin at the leading edge, as well as the effect of the α-actinin-1 construct lacking the actin-binding domain, indicated the critical role played by α-actinin-1 linkage to the actin cytoskeleton in T cell morphology and migration. Figure 7.KIM127+ LFA-1 requires attachment to the actin cytoskeleton via α-actinin-1 for spreading and migration on ICAM-1. (A) Confocal image of a representative HSB-2 cell transfected with α-actinin–GFP (green) and counterstained with Alexa546–phalloidin to detect F-actin (red) following fixation. The level of expression of α-actinin–GFP (green trace) and F-actin staining (red trace) along the length of the polarized T cell (indicated by red arrow in merged image) is recorded. Scale bar=10 μm. (B) Average speed of HSB-2 T cells transfected with either GFP, α-actinin–GFP or ΔN-actinin–GFP (α-actinin-1 mutant missing the actin-binding domain) (mean values of n=3; 36 cells per condition; *P<0.01). (C) Cell tracks and video microscopy images of HSB-2 transfectants as in panel B; 12 cells tracked per condition. Representative experiment of n=3. Scale bar=10 μm. Download figure Download PowerPoint α-Actinin-1 binding to the LFA-1 β2 subunit is necessary for T cell migration The previous experiments showed that α-actinin-1 interacting with F-actin at the leading edge was required for successful T cell migration and that a sub-population of α-actinin-1 is associated with KIM127+ LFA-1. A question was whether the LFA-1/α-actinin-1 association was direct and whether interfering with it would alter aspects of migration that were also altered by manipulating α-actinin-1 itself. To investigate the interaction between LFA-1 and α-actinin-1, we synthesized a cell-permeable peptide corresponding to the α-actinin-1-binding site (H728–S745) (β2-actinin peptide) on the LFA-1 β2 subunit cytoplasmic tail (Pavalko and LaRoche, 1993). In a pull-down assay comparing the β2-actinin peptide (lane 3) with the scrambled control peptide (lane 2), the β2-actinin peptide showed selectivity in binding α-actinin-1 but not talin or vinculin (Figure 8A). Figure 8.Effects of β2-actinin-1 peptide on T cell adhesion, migration and morphology on ICAM-1. (A) Western blot of the following is shown: lane 1, total T cell lysate; lane 2, pull down using a biotin-labelled control peptide; lane 3, pull down using biotin-labelled β2-actinin peptide probed for α-actinin-1, talin and vinculin. (B) Average speed of migration of T cells preincubated without peptide, with control peptide or β2-actinin peptide (mean values of n=3; 24 cells per condition). (C) Video microscopy images of T cells preincubated for 30 min with 50 μg/ml control or β2-actinin peptide. Scale bar=10 μm. Download figure Download PowerPoint T cells were then preincubated with the two peptides to test their effect on migration. The speed of β2-actinin peptide-treated cells was reduced by 87±4%, with the control peptide-treated cells being similar to the untreated cells (Figure 8B). When viewed by real-time low-light microscopy, β2-actinin peptide-treated cells were attached but exhibited dynamic membrane activity, with projections extending and retracting around the perimeter of the cell (Figure 8C). Therefore, the failure to migrate was not due to an inability to generate membrane protrusions (Supplementary Video 4A). Control peptide-treated cells migrated normally (Figure 8C; Supplementary Video 4B). We used IRM to examine in more detail the close contacts with ICAM-1 made by migrating T cells following preincubation with peptide. On untreated (data not shown) and control peptide-treated cells, the lamellar membranes displayed dynamic patterns of intermittent attachment in the direction of cell movement over time, as well as a more uniform darker contact area that followed behind the leading edge (Figure 9A). In contrast, the β2-actinin peptide-treated T cells showed only the dark close contact area, with little evidence for membranes making new contacts over time (Figure 9A). Analysis of cell morphology over 30 s showed that ∼70% of control peptide and untreated cells displayed a defined leading edge comprising lamella and lamellipodium, whereas 50% of the β2-actinin peptide-treated cells had no definite leading edge, and a further 10% showed minimal lamellipodial extension (Figure 9B). The images indicate that exposure of migrating T cells to a peptide designed to disrupt the LFA-1/α-actinin-1 connection interferes with the ability of the cell to generate
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