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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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Multimeric ligand binding to activated LFA-1 but not activation alone induces LFA-1 microclustering. K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (A), αL/β2-mCFP and αL/β2-mYFP (B), or αL-mCFP/β2 and αL-mYFP/β2 + CXCR4 (C) were preincubated with either 1 mM Mn2+, 1 mM Mn2+ + 100 μg/ml sIC-1, IC-Fc/Anti-IgA complex, 1 mM Mn2+ + IC-Fc/Anti-IgA complex, 1 μg/ml SDF-1, 1 μg/ml SDF-1 + 500 μg/ml sIC-1, or 1 μg/ml SDF-1 + IC-Fc/Anti-IgA complex, and were subjected to FRET measurements.
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fig2: Multimeric ligand binding to activated LFA-1 but not activation alone induces LFA-1 microclustering. K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (A), αL/β2-mCFP and αL/β2-mYFP (B), or αL-mCFP/β2 and αL-mYFP/β2 + CXCR4 (C) were preincubated with either 1 mM Mn2+, 1 mM Mn2+ + 100 μg/ml sIC-1, IC-Fc/Anti-IgA complex, 1 mM Mn2+ + IC-Fc/Anti-IgA complex, 1 μg/ml SDF-1, 1 μg/ml SDF-1 + 500 μg/ml sIC-1, or 1 μg/ml SDF-1 + IC-Fc/Anti-IgA complex, and were subjected to FRET measurements.

Mentions: We used ligand-induced activation of LFA-1 to test for a linkage between changes in integrin conformation, specifically separation of α and β subunit cytoplasmic domains within a heterodimer (Kim et al., 2003), and the formation of clusters of LFA-1 molecules through homomeric interaction (Li et al., 2003). Mn2+ has been shown to promote conformational changes in the extracellular domain of LFA-1 that increases its affinity for ICAM-1 (Dransfield et al., 1992). Mn2+-promoted binding of ICAM-1 to LFA-1 induces separation of the αL and β2 cytoplasmic domains (Kim et al., 2003), as confirmed here with intra-heterodimer FRET using αL-mCFP/β2-mYFP transfectants (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200404160/DC1). By contrast, parallel inter-heterodimer αL-αL FRET experiments showed absolutely no microclustering promoted with Mn2+ plus sICAM-1 using αL-mCFP/β2 + αL-mYFP/β2 transfectants (Fig. 2 A, Mn2+ + sIC compared with Mn2+). Similarly, Mn2+ plus sICAM-1 did not promote any β2-β2 FRET in the αL/β2- mCFP + αL/β2-mYFP transfectants (Fig. 2 B). Mn2+ + sICAM-1 also failed to induce macroclustering (Fig. S1 C).


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)

Multimeric ligand binding to activated LFA-1 but not activation alone induces LFA-1 microclustering. K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (A), αL/β2-mCFP and αL/β2-mYFP (B), or αL-mCFP/β2 and αL-mYFP/β2 + CXCR4 (C) were preincubated with either 1 mM Mn2+, 1 mM Mn2+ + 100 μg/ml sIC-1, IC-Fc/Anti-IgA complex, 1 mM Mn2+ + IC-Fc/Anti-IgA complex, 1 μg/ml SDF-1, 1 μg/ml SDF-1 + 500 μg/ml sIC-1, or 1 μg/ml SDF-1 + IC-Fc/Anti-IgA complex, and were subjected to FRET measurements.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172602&req=5

fig2: Multimeric ligand binding to activated LFA-1 but not activation alone induces LFA-1 microclustering. K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (A), αL/β2-mCFP and αL/β2-mYFP (B), or αL-mCFP/β2 and αL-mYFP/β2 + CXCR4 (C) were preincubated with either 1 mM Mn2+, 1 mM Mn2+ + 100 μg/ml sIC-1, IC-Fc/Anti-IgA complex, 1 mM Mn2+ + IC-Fc/Anti-IgA complex, 1 μg/ml SDF-1, 1 μg/ml SDF-1 + 500 μg/ml sIC-1, or 1 μg/ml SDF-1 + IC-Fc/Anti-IgA complex, and were subjected to FRET measurements.
Mentions: We used ligand-induced activation of LFA-1 to test for a linkage between changes in integrin conformation, specifically separation of α and β subunit cytoplasmic domains within a heterodimer (Kim et al., 2003), and the formation of clusters of LFA-1 molecules through homomeric interaction (Li et al., 2003). Mn2+ has been shown to promote conformational changes in the extracellular domain of LFA-1 that increases its affinity for ICAM-1 (Dransfield et al., 1992). Mn2+-promoted binding of ICAM-1 to LFA-1 induces separation of the αL and β2 cytoplasmic domains (Kim et al., 2003), as confirmed here with intra-heterodimer FRET using αL-mCFP/β2-mYFP transfectants (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200404160/DC1). By contrast, parallel inter-heterodimer αL-αL FRET experiments showed absolutely no microclustering promoted with Mn2+ plus sICAM-1 using αL-mCFP/β2 + αL-mYFP/β2 transfectants (Fig. 2 A, Mn2+ + sIC compared with Mn2+). Similarly, Mn2+ plus sICAM-1 did not promote any β2-β2 FRET in the αL/β2- mCFP + αL/β2-mYFP transfectants (Fig. 2 B). Mn2+ + sICAM-1 also failed to induce macroclustering (Fig. S1 C).

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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Related in: MedlinePlus