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The primacy of affinity over clustering in regulation of adhesiveness of the integrin {alpha}L{beta}2.

Kim M, Carman CV, Yang W, Salas A, Springer TA - J. Cell Biol. (2004)

Bottom Line: Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration.Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand.Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering.

View Article: PubMed Central - PubMed

Affiliation: The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.

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Disruption of cytoskeletal constraints does not alter LFA-1 macroclustering. (A–D) K562 cells expressing wild-type αLβ2 were incubated in (A) L15 medium (control); (B) 1 mM Mn2+; (C) 1 μM PMA; or (D) 1 μM cytochalasin D for 30 min at 37°C. After fixation, cells were stained with Cy3-conjugated TS2/4 mAb. (E) Cells were incubated with Cy3-TS2/4 mAb (10 μg/ml) together with purified anti–mouse IgG (10 μg/ml) at 37°C for 30 min followed by fixation. All cells (A–E) were then plated on coverslips and subjected to confocal microscopy. Center panels depict a threefold magnification of the boxed regions shown in the left panels. Three-dimensional histograms of fluorescence intensity and cell surface distribution are shown in the right panels.
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fig4: Disruption of cytoskeletal constraints does not alter LFA-1 macroclustering. (A–D) K562 cells expressing wild-type αLβ2 were incubated in (A) L15 medium (control); (B) 1 mM Mn2+; (C) 1 μM PMA; or (D) 1 μM cytochalasin D for 30 min at 37°C. After fixation, cells were stained with Cy3-conjugated TS2/4 mAb. (E) Cells were incubated with Cy3-TS2/4 mAb (10 μg/ml) together with purified anti–mouse IgG (10 μg/ml) at 37°C for 30 min followed by fixation. All cells (A–E) were then plated on coverslips and subjected to confocal microscopy. Center panels depict a threefold magnification of the boxed regions shown in the left panels. Three-dimensional histograms of fluorescence intensity and cell surface distribution are shown in the right panels.

Mentions: To determine whether actin-disrupting agents or PMA directly alter LFA-1 distribution patterns, we conducted confocal (Fig. 4) and FRET (Fig. 5) microscopy studies. Cross-linking cell surface LFA-1 with primary and secondary antibodies efficiently promoted LFA-1 macroclustering; however, treatment with cytochalasin D, latrunculin A, and PMA did not (Fig. 4 and unpublished data). Some alteration in the shape of PMA-treated cells was attributable to the ability of PMA to induce bleb formation in K562 cells (Osada et al., 1988), as confirmed by differential interference contrast (DIC) imaging (unpublished data). The amount of FRET between LFA-1 heterodimers in PMA- and cytochalasin D–treated cells measured either between αL subunits (Fig. 5 A) or between β2 subunits (Fig. 5 B) was similar to that in untreated cells (Fig. 1, C and F). This demonstrated an absence of significant microclustering induced by treatment with PMA or cytochalasin D. Thus, release of cytoskeletal constraints enables robust adhesion to ICAM-1 substrates, but does not drive ligand-independent integrin macro- or microclustering.


The primacy of affinity over clustering in regulation of adhesiveness of the integrin {alpha}L{beta}2.

Kim M, Carman CV, Yang W, Salas A, Springer TA - J. Cell Biol. (2004)

Disruption of cytoskeletal constraints does not alter LFA-1 macroclustering. (A–D) K562 cells expressing wild-type αLβ2 were incubated in (A) L15 medium (control); (B) 1 mM Mn2+; (C) 1 μM PMA; or (D) 1 μM cytochalasin D for 30 min at 37°C. After fixation, cells were stained with Cy3-conjugated TS2/4 mAb. (E) Cells were incubated with Cy3-TS2/4 mAb (10 μg/ml) together with purified anti–mouse IgG (10 μg/ml) at 37°C for 30 min followed by fixation. All cells (A–E) were then plated on coverslips and subjected to confocal microscopy. Center panels depict a threefold magnification of the boxed regions shown in the left panels. Three-dimensional histograms of fluorescence intensity and cell surface distribution are shown in the right panels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172602&req=5

fig4: Disruption of cytoskeletal constraints does not alter LFA-1 macroclustering. (A–D) K562 cells expressing wild-type αLβ2 were incubated in (A) L15 medium (control); (B) 1 mM Mn2+; (C) 1 μM PMA; or (D) 1 μM cytochalasin D for 30 min at 37°C. After fixation, cells were stained with Cy3-conjugated TS2/4 mAb. (E) Cells were incubated with Cy3-TS2/4 mAb (10 μg/ml) together with purified anti–mouse IgG (10 μg/ml) at 37°C for 30 min followed by fixation. All cells (A–E) were then plated on coverslips and subjected to confocal microscopy. Center panels depict a threefold magnification of the boxed regions shown in the left panels. Three-dimensional histograms of fluorescence intensity and cell surface distribution are shown in the right panels.
Mentions: To determine whether actin-disrupting agents or PMA directly alter LFA-1 distribution patterns, we conducted confocal (Fig. 4) and FRET (Fig. 5) microscopy studies. Cross-linking cell surface LFA-1 with primary and secondary antibodies efficiently promoted LFA-1 macroclustering; however, treatment with cytochalasin D, latrunculin A, and PMA did not (Fig. 4 and unpublished data). Some alteration in the shape of PMA-treated cells was attributable to the ability of PMA to induce bleb formation in K562 cells (Osada et al., 1988), as confirmed by differential interference contrast (DIC) imaging (unpublished data). The amount of FRET between LFA-1 heterodimers in PMA- and cytochalasin D–treated cells measured either between αL subunits (Fig. 5 A) or between β2 subunits (Fig. 5 B) was similar to that in untreated cells (Fig. 1, C and F). This demonstrated an absence of significant microclustering induced by treatment with PMA or cytochalasin D. Thus, release of cytoskeletal constraints enables robust adhesion to ICAM-1 substrates, but does not drive ligand-independent integrin macro- or microclustering.

Bottom Line: Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration.Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand.Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering.

View Article: PubMed Central - PubMed

Affiliation: The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.

Show MeSH
Related in: MedlinePlus