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A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes.

Smith A, Carrasco YR, Stanley P, Kieffer N, Batista FD, Hogg N - J. Cell Biol. (2005)

Bottom Line: Talin is essential for the stability and formation of the LFA-1 zone.Disruption of the talin-integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1.This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the "focal zone."

View Article: PubMed Central - PubMed

Affiliation: Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, England, UK.

ABSTRACT
Cells such as fibroblasts and endothelial cells migrate through the coordinated responses of discrete integrin-containing focal adhesions and complexes. In contrast, little is known about the organization of integrins on the highly motile T lymphocyte. We have investigated the distribution, activity, and cytoskeletal linkage of the integrin lymphocyte function associated antigen-1 (LFA-1) on human T lymphocytes migrating on endothelial cells and on ligand intercellular adhesion molecule-1 (ICAM-1). The pattern of total LFA-1 varies from low expression in the lamellipodia to high expression in the uropod. However, high affinity, clustered LFA-1 is restricted to a mid-cell zone that remains stable over time and over a range of ICAM-1 densities. Talin is essential for the stability and formation of the LFA-1 zone. Disruption of the talin-integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1. This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the "focal zone."

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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.
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fig6: 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.

Mentions: The build-up of LFA-1 in the focal zone suggested that the mobility of LFA-1 must be regulated, probably by cytoskeletal attachment. To test this idea further, we compared the dynamics of total LFA-1 and high affinity (mAb 24 bound) LFA-1 by FRAP of an area within the focal zone. Fluorescent LFA-1 moved into the bleached region but did not completely replace bleached LFA-1 during the course of the experiment (Fig. 6, A and C, green). Locking LFA-1 into the high affinity conformation with mAb 24 resulted in the further inhibition of membrane mobility in the focal zone as seen by the lack of recovery in fluorescence (Fig. 6, B and C, green). Quantification of the mobile fraction of LFA-1 located in the focal zone shows it to be reduced from 38.8 to 6.2%, indicating that high affinity LFA-1 is under cytoskeletal constraint. These results provide evidence of a dynamic relationship between high affinity LFA-1 linked to the cytoskeleton and a mobile fraction of LFA-1 within the zone (Table I).


A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes.

Smith A, Carrasco YR, Stanley P, Kieffer N, Batista FD, Hogg N - J. Cell Biol. (2005)

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.
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fig6: 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.
Mentions: The build-up of LFA-1 in the focal zone suggested that the mobility of LFA-1 must be regulated, probably by cytoskeletal attachment. To test this idea further, we compared the dynamics of total LFA-1 and high affinity (mAb 24 bound) LFA-1 by FRAP of an area within the focal zone. Fluorescent LFA-1 moved into the bleached region but did not completely replace bleached LFA-1 during the course of the experiment (Fig. 6, A and C, green). Locking LFA-1 into the high affinity conformation with mAb 24 resulted in the further inhibition of membrane mobility in the focal zone as seen by the lack of recovery in fluorescence (Fig. 6, B and C, green). Quantification of the mobile fraction of LFA-1 located in the focal zone shows it to be reduced from 38.8 to 6.2%, indicating that high affinity LFA-1 is under cytoskeletal constraint. These results provide evidence of a dynamic relationship between high affinity LFA-1 linked to the cytoskeleton and a mobile fraction of LFA-1 within the zone (Table I).

Bottom Line: Talin is essential for the stability and formation of the LFA-1 zone.Disruption of the talin-integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1.This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the "focal zone."

View Article: PubMed Central - PubMed

Affiliation: Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, England, UK.

ABSTRACT
Cells such as fibroblasts and endothelial cells migrate through the coordinated responses of discrete integrin-containing focal adhesions and complexes. In contrast, little is known about the organization of integrins on the highly motile T lymphocyte. We have investigated the distribution, activity, and cytoskeletal linkage of the integrin lymphocyte function associated antigen-1 (LFA-1) on human T lymphocytes migrating on endothelial cells and on ligand intercellular adhesion molecule-1 (ICAM-1). The pattern of total LFA-1 varies from low expression in the lamellipodia to high expression in the uropod. However, high affinity, clustered LFA-1 is restricted to a mid-cell zone that remains stable over time and over a range of ICAM-1 densities. Talin is essential for the stability and formation of the LFA-1 zone. Disruption of the talin-integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1. This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the "focal zone."

Show MeSH
Related in: MedlinePlus