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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 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.
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fig5: 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.

Mentions: Next, we wanted to explore the activation status of LFA-1 on the migrating T cell. To locate LFA-1 in the high affinity conformation, we used Fab′ fragments of mAb 24, which selectively recognizes high affinity LFA-1 (Dransfield and Hogg, 1989; Hogg et al., 2002). When added to migrating T cells, mAb 24 immediately localized to the focal zone (Fig. 5 A, compare ICAM-1 [red] with mAb 24 [green]), providing evidence that this region contained high affinity LFA-1. Localization of high affinity LFA-1 to the focal zone was also a feature of T cells migrating on target cells expressing ICAM-1-GFP (Fig. 5 B). In contrast, no mAb 24 binding was displayed by the uropod (Fig. 5 A, DIC) nor was it detectable in the lamellipodia, even after long exposures to this mAb (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200412032/DC1). Therefore, it is likely that the leading edge adhesions and uropod do not involve high affinity LFA-1 or at least only at very low levels.


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 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.
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Related In: Results  -  Collection

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fig5: 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.
Mentions: Next, we wanted to explore the activation status of LFA-1 on the migrating T cell. To locate LFA-1 in the high affinity conformation, we used Fab′ fragments of mAb 24, which selectively recognizes high affinity LFA-1 (Dransfield and Hogg, 1989; Hogg et al., 2002). When added to migrating T cells, mAb 24 immediately localized to the focal zone (Fig. 5 A, compare ICAM-1 [red] with mAb 24 [green]), providing evidence that this region contained high affinity LFA-1. Localization of high affinity LFA-1 to the focal zone was also a feature of T cells migrating on target cells expressing ICAM-1-GFP (Fig. 5 B). In contrast, no mAb 24 binding was displayed by the uropod (Fig. 5 A, DIC) nor was it detectable in the lamellipodia, even after long exposures to this mAb (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200412032/DC1). Therefore, it is likely that the leading edge adhesions and uropod do not involve high affinity LFA-1 or at least only at very low levels.

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