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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 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.
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fig3: 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.

Mentions: To investigate total LFA-1 expression, we stained migrating T cells with Alexa 488–conjugated nonblocking anti–LFA-1 Fab′ fragments as in Fig. 1 A. The distribution of LFA-1 was found to be substantially increased in two cellular locations: toward the mid-cell region, where it overlapped with the high levels of ICAM-1 in the focal zone, and in the uropod, where concentrated ICAM-1 was not observed (Fig. 3 A). Serial sections through a migrating T cell revealed that although LFA-1 was concentrated at the level of the bilayer, it also surrounded the entire surface of the cell with high levels in the uropod (Fig. 3 B). This distinctive pattern of LFA-1 distribution is in contrast to other membrane proteins such as CD3, which is evenly expressed over the entire membrane, as well as CD43 and ICAM-3, which are concentrated in the uropod (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412032/DC1).


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171377&req=5

fig3: 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.
Mentions: To investigate total LFA-1 expression, we stained migrating T cells with Alexa 488–conjugated nonblocking anti–LFA-1 Fab′ fragments as in Fig. 1 A. The distribution of LFA-1 was found to be substantially increased in two cellular locations: toward the mid-cell region, where it overlapped with the high levels of ICAM-1 in the focal zone, and in the uropod, where concentrated ICAM-1 was not observed (Fig. 3 A). Serial sections through a migrating T cell revealed that although LFA-1 was concentrated at the level of the bilayer, it also surrounded the entire surface of the cell with high levels in the uropod (Fig. 3 B). This distinctive pattern of LFA-1 distribution is in contrast to other membrane proteins such as CD3, which is evenly expressed over the entire membrane, as well as CD43 and ICAM-3, which are concentrated in the uropod (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412032/DC1).

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