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Comparative Study
. 2008 Jan 9;27(1):62-75.
doi: 10.1038/sj.emboj.7601959. Epub 2007 Dec 13.

Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell

Paula Stanley  1 , Andrew Smith, Alison McDowall, Alastair Nicol, Daniel Zicha, Nancy Hogg
Affiliations
Comparative Study

Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell

Paula Stanley et al. EMBO J. .

Abstract

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 alpha-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.

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Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
Figure 5
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).
Figure 6
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).
Figure 7
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.
Figure 8
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.
Figure 9
Figure 9
Effect of β2-actinin-1 peptide on the IRM image of migrating T cells. (A) IRM images of representative T cells treated with control scrambled peptide or β2-actinin peptide for 30 min before migrating on ICAM-1. The three frames show images recorded at 0, 6 and 12 s. Images are representative of 40 cells per treatment group (n=3). Scale bar=5 μm. (B) Analysis of IRM images of T cells treated with β2-actinin or control peptide. The IRM ‘bright' image for each cell was scored as extensive (lamella+), partial (intermediate) or absence of obvious lamella/lamellipodial region (n=40 for each group in two experiments).
Figure 10
Figure 10
Effect of β2-actinin peptide on the IRM image of migrating T cells following treatment with KIM127. IRM images are shown of T cells that are pretreated with 10 μg/ml KIM127, resulting in KIM127-stabilized adhesions (see dark contact areas). The cells are then treated with either β2-actinin or control peptide and the IRM image recorded after 200 s. The β2-actinin peptide caused the KIM127 stabilized adhesions to diminish, whereas the control peptide had no effect. Scale bar=10 μm.
Figure 11
Figure 11
FRET analysis showing association between LFA-1 and α-actinin-1 at the leading edge of HSB-2 T cells. (A) AdFRET profile showing fluorescence intensity of acceptor Alexa546-labelled α-actinin-1 and donor GFP-LFA-1 for five frames before and five frames after photobleaching of the acceptor (n=10 for each zone tested). The increase in fluorescence intensity of donor LFA-1 occurs at the leading edge in the lamella of the migrating T cell but not in the focal zone or uropod. (B) AdFRET profiles at the leading edge were determined as in panel A for cells treated with β2-actinin peptide, control peptide or no peptide. No FRET was seen with addition of β2-actinin peptide, showing that LFA-1 does associate with α-actinin-1 through the β2 cytoplasmic tail sequence covered by the β2-actinin peptide.
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