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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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Experimental design and validation of inter-heterodimer FRET microclustering assay. (A) A hypothetical model (Li et al., 2003) for integrin clustering. Cells expressing heterodimers with either the α (shown) or β subunits tagged with both mCFP and mYFP will exhibit FRET only when heterodimers are brought into close proximity (< 100 Å). (B) Schematic (top) and model curves (bottom) for FRET behavior under nonclustered and microclustered conditions (Kenworthy et al., 2000; Zacharias et al., 2002). See Results. (C–G) Inter-heterodimer FRET and acceptor intensities for individual ROIs from K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (C and E), αL-CFP/β2 and αL-YFP/β2 (D), or αL/β2-mCFP and αL/β2-mYFP (F and G) were fit to  (red curves) using the Lineweaver-Burke equation as described in Materials and methods. Where indicated, cell surface LFA-1 was cross-linked by preincubation with either 10 μg/ml of TS2/4 mAb to αL (E) or CBR LFA-1/7 mAb to β2 (G) and secondary, purified goat anti–mouse antibody (10 μg/ml) for 30 min at 37°C. Representative confocal images, depicting the YFP signal from selected experiments (C–E), are shown below the graphs.
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fig1: Experimental design and validation of inter-heterodimer FRET microclustering assay. (A) A hypothetical model (Li et al., 2003) for integrin clustering. Cells expressing heterodimers with either the α (shown) or β subunits tagged with both mCFP and mYFP will exhibit FRET only when heterodimers are brought into close proximity (< 100 Å). (B) Schematic (top) and model curves (bottom) for FRET behavior under nonclustered and microclustered conditions (Kenworthy et al., 2000; Zacharias et al., 2002). See Results. (C–G) Inter-heterodimer FRET and acceptor intensities for individual ROIs from K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (C and E), αL-CFP/β2 and αL-YFP/β2 (D), or αL/β2-mCFP and αL/β2-mYFP (F and G) were fit to (red curves) using the Lineweaver-Burke equation as described in Materials and methods. Where indicated, cell surface LFA-1 was cross-linked by preincubation with either 10 μg/ml of TS2/4 mAb to αL (E) or CBR LFA-1/7 mAb to β2 (G) and secondary, purified goat anti–mouse antibody (10 μg/ml) for 30 min at 37°C. Representative confocal images, depicting the YFP signal from selected experiments (C–E), are shown below the graphs.

Mentions: To quantitatively measure integrin clustering on the cell surface at the scale of molecular interactions, i.e., microclustering, we developed FRET-based assays. Nondimerizing or “monomeric” (m) mutants (Zacharias et al., 2002) of CFP (mCFP) and YFP (mYFP) were fused to the COOH termini of the αL and β2 subunit cytoplasmic domains (Fig. 1 A). To measure inter-heterodimer distances, K562 cells were transiently transfected with αL-mCFP, αL-mYFP, and wild-type β2, generating cell surface expression of αL-mCFP/β2 and αL-mYFP/β2 in approximately equal amounts. The redistribution of integrin heterodimers into microclusters, in which the αL subunits come within 100 Å of each other, should result in detectable CFP/YFP FRET. Similarly, cells were transfected with wild-type αL, β2-mCFP and β2-mYFP, such that FRET would measure proximity of β2 subunits of separate integrin heterodimers.


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)

Experimental design and validation of inter-heterodimer FRET microclustering assay. (A) A hypothetical model (Li et al., 2003) for integrin clustering. Cells expressing heterodimers with either the α (shown) or β subunits tagged with both mCFP and mYFP will exhibit FRET only when heterodimers are brought into close proximity (< 100 Å). (B) Schematic (top) and model curves (bottom) for FRET behavior under nonclustered and microclustered conditions (Kenworthy et al., 2000; Zacharias et al., 2002). See Results. (C–G) Inter-heterodimer FRET and acceptor intensities for individual ROIs from K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (C and E), αL-CFP/β2 and αL-YFP/β2 (D), or αL/β2-mCFP and αL/β2-mYFP (F and G) were fit to  (red curves) using the Lineweaver-Burke equation as described in Materials and methods. Where indicated, cell surface LFA-1 was cross-linked by preincubation with either 10 μg/ml of TS2/4 mAb to αL (E) or CBR LFA-1/7 mAb to β2 (G) and secondary, purified goat anti–mouse antibody (10 μg/ml) for 30 min at 37°C. Representative confocal images, depicting the YFP signal from selected experiments (C–E), are shown below the graphs.
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Related In: Results  -  Collection

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fig1: Experimental design and validation of inter-heterodimer FRET microclustering assay. (A) A hypothetical model (Li et al., 2003) for integrin clustering. Cells expressing heterodimers with either the α (shown) or β subunits tagged with both mCFP and mYFP will exhibit FRET only when heterodimers are brought into close proximity (< 100 Å). (B) Schematic (top) and model curves (bottom) for FRET behavior under nonclustered and microclustered conditions (Kenworthy et al., 2000; Zacharias et al., 2002). See Results. (C–G) Inter-heterodimer FRET and acceptor intensities for individual ROIs from K562 cells expressing αL-mCFP/β2 and αL-mYFP/β2 (C and E), αL-CFP/β2 and αL-YFP/β2 (D), or αL/β2-mCFP and αL/β2-mYFP (F and G) were fit to (red curves) using the Lineweaver-Burke equation as described in Materials and methods. Where indicated, cell surface LFA-1 was cross-linked by preincubation with either 10 μg/ml of TS2/4 mAb to αL (E) or CBR LFA-1/7 mAb to β2 (G) and secondary, purified goat anti–mouse antibody (10 μg/ml) for 30 min at 37°C. Representative confocal images, depicting the YFP signal from selected experiments (C–E), are shown below the graphs.
Mentions: To quantitatively measure integrin clustering on the cell surface at the scale of molecular interactions, i.e., microclustering, we developed FRET-based assays. Nondimerizing or “monomeric” (m) mutants (Zacharias et al., 2002) of CFP (mCFP) and YFP (mYFP) were fused to the COOH termini of the αL and β2 subunit cytoplasmic domains (Fig. 1 A). To measure inter-heterodimer distances, K562 cells were transiently transfected with αL-mCFP, αL-mYFP, and wild-type β2, generating cell surface expression of αL-mCFP/β2 and αL-mYFP/β2 in approximately equal amounts. The redistribution of integrin heterodimers into microclusters, in which the αL subunits come within 100 Å of each other, should result in detectable CFP/YFP FRET. Similarly, cells were transfected with wild-type αL, β2-mCFP and β2-mYFP, such that FRET would measure proximity of β2 subunits of separate integrin heterodimers.

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