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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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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.
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fig7: 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.

Mentions: Because the FRAP studies indicated that high affinity LFA-1 was not freely mobile in the membrane, we explored which cytoskeletal proteins might be associated with LFA-1 in the focal zone. Talin was considered to be a prime candidate, as it has previously been reported to bind directly to β2 integrin (Pavalko and LaRoche, 1993; Sampath et al., 1998) and to activate LFA-1 (Kim et al., 2003). The T cell line HSB-2 stably expressing the GFP-tagged β2 subunit of LFA-1 (β2-GFP) was plated onto ICAM-1 and allowed to migrate before fixation and counterstaining for talin. The distribution of β2-GFP was in accordance with the LFA-1 pattern reported in Fig. 3 for T cells migrating on ICAM-1 (Fig. 7 A). With regard to talin, the highest level localized to the focal zone (Fig. 7 A, a vs. b; and Fig. 7 A, d) and a composite image in the y-z axis showed it to be concentrated at the interface of LFA-1 interacting with ICAM-1 (Fig. 7 A, c and e). This was in contrast to other intracellular proteins involved in T cell polarization and migration such as the uropod-located ezrin and the lamellipodia-associated Rac-1 and Vav-1 (Fig. S2). Therefore, talin is specifically associated with the focal zone where high affinity LFA-1 is located. To further characterize this association, we transiently transfected HSB-2 cells with a talin head domain–GFP construct (Tremuth et al., 2004) and allowed the cells to migrate on the ICAM-1–embedded lipid bilayer. Talin head–GFP localized to the region of concentrated ICAM-1 corresponding to the focal zone (Fig. 7 B and Fig. S3).


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)

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.
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fig7: 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.
Mentions: Because the FRAP studies indicated that high affinity LFA-1 was not freely mobile in the membrane, we explored which cytoskeletal proteins might be associated with LFA-1 in the focal zone. Talin was considered to be a prime candidate, as it has previously been reported to bind directly to β2 integrin (Pavalko and LaRoche, 1993; Sampath et al., 1998) and to activate LFA-1 (Kim et al., 2003). The T cell line HSB-2 stably expressing the GFP-tagged β2 subunit of LFA-1 (β2-GFP) was plated onto ICAM-1 and allowed to migrate before fixation and counterstaining for talin. The distribution of β2-GFP was in accordance with the LFA-1 pattern reported in Fig. 3 for T cells migrating on ICAM-1 (Fig. 7 A). With regard to talin, the highest level localized to the focal zone (Fig. 7 A, a vs. b; and Fig. 7 A, d) and a composite image in the y-z axis showed it to be concentrated at the interface of LFA-1 interacting with ICAM-1 (Fig. 7 A, c and e). This was in contrast to other intracellular proteins involved in T cell polarization and migration such as the uropod-located ezrin and the lamellipodia-associated Rac-1 and Vav-1 (Fig. S2). Therefore, talin is specifically associated with the focal zone where high affinity LFA-1 is located. To further characterize this association, we transiently transfected HSB-2 cells with a talin head domain–GFP construct (Tremuth et al., 2004) and allowed the cells to migrate on the ICAM-1–embedded lipid bilayer. Talin head–GFP localized to the region of concentrated ICAM-1 corresponding to the focal zone (Fig. 7 B and Fig. S3).

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