IRM reveals the areas of direct contact between migrating T cells and ICAM-1. (A) The DIC image compared with the IRM image and the ICAM-1 binding focal zone (red). The IRM image identifies the total area of the T cell in direct contact with ICAM-1 in the bilayer and demonstrates intermittent, close, and lack of attachments in lamellipodia, focal zone, and uropod, respectively. Double-sided arrow, closest attachment to the substrate; black arrow, the leading edge; white arrow, the uropod of the migrating T cell. Overlaying the ICAM-1 and IRM images shows that the area of uniform attachment colocalizes with the focal zone. (B) Quantification of the area in contact with the bilayer compared with the area of the focal zone (n = 10; ±SD). All images were taken with a 63× oil immersion objective. Bars, 10 μm.

The mobility of total and high affinity LFA-1 within the focal zone. (A) T cells were labeled with nonfunction blocking Alexa 488–conjugated anti–LFA-1 Fab′ and allowed to attach and migrate on the ICAM-1 bilayer. An area within the zone of ICAM-1 was photobleached (circled; 2 μm diam) at time 0 and fluorescence recovery was recorded over 60 s. LFA-1 (green) showed partial recovery, whereas ICAM-1 (red) rapidly recovers and is complete by 30 s. The boxed region shows the T cell region enlarged in subsequent images. (B) T cells were allowed to attach and migrate on the ICAM-1 bilayer in the presence of Alexa 488–labeled mAb 24 Fab′. An area within the zone of ICAM-1 was photobleached at time 0 and fluorescence recovery was recorded over 120 s. High affinity LFA-1 (green) showed no recovery, whereas ICAM-1 (red) partially recovers by 120 s. (C) Quantification of the recovery in the focal zone of LFA-1 (closed triangle), high affinity LFA-1 (+ mAb 24) (shaded triangle), ICAM-1 (closed square), ICAM-1 (+ mAb 24) (shaded square), and ICAM-1 within the bilayer as a control (closed circle) (n = 8; ±SD). All images were taken with a 63× oil immersion objective. Bars, 10 μm.

Confocal image of a T cell migrating in the presence of 5 mM MgCl₂ on a target cell expressing ICAM-1. T cells labeled with Alexa 488 nonblocking anti–LFA-1 Fabs and migrating on TNF-α–activated HUVECs. (A) Phase images of the pattern of migration of four T cells (1–4) over 3 min. (B) False color images of the same four T cells showing expression levels of LFA-1 (highest [white] to lowest [blue]). The asterisk denotes the leading edge of each cell. (C) T cell (white outline) migrating on ICAM-1–transfected COS-7 cells (the asterisk denotes the T cell leading edge). (a) T cell labeled with anti–LFA-1 mAb (red). (b) COS-7 cells expressing ICAM-1-GFP (green). (c) Merge of the two images in panels a and b. Colocalization of LFA-1 and ICAM-1 can be observed in the mid-cell region. All images were taken with a 63× oil immersion objective. Bars, 10 μm. (d) Quantitation of ICAM-1 density in focal zone (black bar) compared with lamellar region (gray bar) on migrating T cells (n = 9; ±SD). *, P < 0.001.

LFA-1 surface distribution on T cells migrating on ICAM-1. Alexa 488–labeled nonfunctional blocking anti–LFA-1 Fab′ is used to identify surface distribution of LFA-1 without cross-linking or affecting ICAM-1 binding activity. (A) Distribution of ICAM-1 (red) and LFA-1 (green) on a migrating T cell at the lipid bilayer level. The merge image demonstrates colocalization of LFA-1 and ICAM-1 in the mid-cell region. (B) Confocal z-stack (each slice = 1 μm) through a T cell migrating on ICAM-1. LFA-1 (green) is most intense at the level of the lipid bilayer in the mid-cell region, with weak expression in the lamellipodia. LFA-1 is visible over the entire surface, with a pattern of increased intensity toward the trailing edge and the uropod. (C) Distribution of LFA-1 (green) and concentrated ICAM-1 (red) was recorded over 180 s after initial contact with ICAM-1 in the lipid bilayer. The top panels show DIC images of a T cell polarizing and migrating. The bottom image panels illustrate the merged LFA-1 and ICAM-1 signals. The arrows (Merge) are split into red (bilayer only) and black (cell and bilayer) and show the regions where the profile data (bottom) was calculated. The arrow tip is located at the trailing edge. The graphs depict the fold increase of LFA-1 compared with the single plasma membrane level in the lamellipodia (green) and ICAM-1 compared with the bilayer level (red) over the entire T cell between 30–180 s after initial contact. All images were taken with 63× oil immersion objective. Bars, 10 μm.

The location of high affinity LFA-1 on T cells migrating on ICAM-1. (A) A T cell migrating on ICAM-1 (red) and labeled with mAb 24 Fab′ (green) showing the distribution of the high affinity LFA-1. The merged image shows the colocalization of mAb 24–bound LFA-1 with the ICAM-1 within the focal zone. (B) T cells (white outline; asterisk denotes the leading edge) migrating on ICAM-1-GFP–transfected COS-7 cells in the presence of Alexa 546 Fab′ of mAb 24 (10 μg/ml) for 120 s before fixation. (C) A T cell migrating on ICAM-1 after the addition of mAb 24 at 0 s shows a build up of ICAM-1 restricted to the focal zone over 80 s. (D) Quantification of the ICAM-1 build up after mAb 24 addition reveals a linear relationship over 112 s (r² = 0.93), resulting in the ICAM-1 density rising from ∼400 to ∼1,500 molecules/μm², equating to ∼10 molecules/μm²/s (n = 5; ±SD). (E) Prolonged exposure to mAb 24 (>300 s) results in the extension of the focal zone into the uropod and an elongated morphology. In comparison, the ICAM-1 levels within the lamellipodia remain the same as in the bilayer. All images were taken with a 63× oil immersion objective. Bars, 10 μm.

Structure and stability of the ICAM-1 binding zone on migrating T cells. Human T cells were added to ICAM-1–containing planar lipid bilayers (175 molecules/μm²) in the presence of 5 mM MgCl₂ at 37°C. (A) The location of a concentrated zone of ICAM-1 (red) on a migrating T cell. (B) A T cell migrating over time displays the stability of the zone of ICAM-1 binding in relation to its intensity and cellular position. (C) The total amount of ICAM-1 within the zone of migrating T cells was calculated at time 0 (equals 100%) and the relative ICAM-1 levels were recorded at intervals over a period of 130 s. All results are expressed as an average of 10 cells ± SD. (D) The lack of ICAM-1 incorporation in the bilayer resulted in the failure of T cell attachment and polarization. Bars, 10 μm. (E) The density of ICAM-1 in the zone decreases as the ICAM-1 level in the lipid bilayer decreases (r² = 0.9677). (F) The area occupied by the zone of ICAM-1 shows stability over a range of ICAM-1 concentrations (175 to 44 molecules/μm²) and then declines by 22 molecules/μm². (G) The migration speed is constant between 175 and 44 molecules/μm² and reduces significantly (P < 0.01) below this level. E–G were composed from values obtained from the same experiments (n = 5–7; ±SD). All images were taken with a 63× oil immersion objective. *, P < 0.01.

LFA-1 and talin colocalize to the focal zone, and effects on migration upon disruption of talin stability. (A) The HSB-2 T cell line expressing β2-GFP migrating on ICAM-1 was fixed and stained for talin. (a) Confocal image of LFA-1-GFP distribution was taken at the coverslip level (asterisk denotes leading edge). (b) An x-y projection of endogenous talin. (c) A y-z projection stained for talin. (d) An x-y projection of the merged LFA-1 (green) and talin (red) images. (e) A y-z projection of the merged LFA-1 (green) and talin (red) images. (B) The HSB-2 T cell line transfected with talin head domain–GFP (green) and allowed to migrate on ICAM-1 (red) after the addition of mAb 24. The merged image shows the colocalization of talin with concentrated ICAM-1. All images were taken with a 63× oil immersion objective. Bars, 10 μm. (C) HSB-2 cells were transfected with siRNA, and the effects on focal zone formation were calculated for talin knock-down and a control sequence. The knock-down of talin had a significant (P < 0.001) reduction in ICAM-1 accumulation in the focal zone compared with control siRNA (n = 14 control [shaded circle], mean value [open circle]; n = 24 talin [closed triangle], mean value [open triangle]). (D) Western blot analysis of talin knock-down in HSB-2 control cells (lane 1) and talin knock-down cells (lane 2) probed for talin and two control cytoskeletal proteins (α-actinin and α-tubulin). (E) T cell ICAM-1 migration assay using talin knock-down cells (n = 40) compared with control cells (n = 40). Results are expressed as a percentage of control cells ± SEM (*, P < 0.05). (F) The migratory tracks produced by T cells migrating on ICAM-1. a, control cells (n = 40); b, talin knock-down cells (n = 40). The red circle indicates a distance of 20 μm from the original position. (G) T cell activated with 5 mM MgCl₂ were plated onto immobilized ICAM-1 in the presence of 30 μg/ml PIPKIγ peptide (n = 60), PIPKIγ control (n = 54), or untreated (n = 26) and tracked for 20 min. Results are expressed as a percentage of untreated cells ± SEM (*, P < 0.05). (H) The effect on T cell migration and focal zone formation in the presence of the PIPKIγ peptide. T cells were allowed to attach and migrate on ICAM-1 lipid bilayers in the presence of 5 mM Mg²⁺. The PIPKIγ peptide (30 μg/ml) was injected (0 s) and the T cell followed for 600 s. The image represents the DIC image overlaid with the ICAM-1 (red) distribution.