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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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Schematic of integrin affinity, diffusivity, and clustering. Inside-out signaling alters integrin affinity and cytoskeletal disruption alters integrin diffusity. How these regulate binding to ligand and the consequences for micro- and macroclustering are shown. Note that the additional effect of integrin redistribution in polarized cells was not studied here and is not shown.
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fig9: Schematic of integrin affinity, diffusivity, and clustering. Inside-out signaling alters integrin affinity and cytoskeletal disruption alters integrin diffusity. How these regulate binding to ligand and the consequences for micro- and macroclustering are shown. Note that the additional effect of integrin redistribution in polarized cells was not studied here and is not shown.

Mentions: In this paper we have addressed whether LFA-1 clustering occurs independently of, or as a consequence of, ligand binding, and whether clustering can be induced by alterations in integrin conformation. Before the discovery of the structural basis (Shimaoka et al., 2003) for intermediate affinity states of LFA-1 (Lollo et al., 1993; Shimaoka et al., 2003), clustering was widely accepted as the mechanism of action for many agents such as PMA and actin microfilament disrupters that, in contrast to Mn2+, induce adhesiveness of LFA-1 but not detectable high affinity binding to ICAM-1 (Dransfield et al., 1992). Because of the perceived need to explain this disparity, it was generally accepted prima facie that clusters would be preformed. Our analysis of both macro- and microclustering demonstrate that, in the absence of either a multivalent ligand on a substrate or homotypic cell aggregation, PMA, cytochalasin D, latrunculin A, and chemokine treatments do not alter integrin distribution patterns despite their ability to promote significant adhesion (Fig. 9). These agents failed to increase FRET between neighboring αL-mCFP/β2 and αL-mYFP/β2 molecules or between neighboring αL/β2-mCFP and αL/β2-mYFP molecules. This agrees with our previous observation that stimulation with chemokines, Mn2+ plus sICAM-1, and PMA induced separation between the cytoplasmic domains of the αL and β2 subunits as shown by a decrease in FRET between αL-mCFP/β2-mYFP in individual LFA-1 heterodimers (Kim et al., 2003). Previous experiments designed to assess integrin macroclustering were often performed under conditions that promote robust homotypic cell aggregation; no effort was made to prevent homotypic adhesion, the effect of homotypic adhesion or a preincubation period in monodisperse cell suspension on cluster formation was not tested, and somtimes it was emphasized that after activation cells were fixed immediately before preparation for microscopy.


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)

Schematic of integrin affinity, diffusivity, and clustering. Inside-out signaling alters integrin affinity and cytoskeletal disruption alters integrin diffusity. How these regulate binding to ligand and the consequences for micro- and macroclustering are shown. Note that the additional effect of integrin redistribution in polarized cells was not studied here and is not shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172602&req=5

fig9: Schematic of integrin affinity, diffusivity, and clustering. Inside-out signaling alters integrin affinity and cytoskeletal disruption alters integrin diffusity. How these regulate binding to ligand and the consequences for micro- and macroclustering are shown. Note that the additional effect of integrin redistribution in polarized cells was not studied here and is not shown.
Mentions: In this paper we have addressed whether LFA-1 clustering occurs independently of, or as a consequence of, ligand binding, and whether clustering can be induced by alterations in integrin conformation. Before the discovery of the structural basis (Shimaoka et al., 2003) for intermediate affinity states of LFA-1 (Lollo et al., 1993; Shimaoka et al., 2003), clustering was widely accepted as the mechanism of action for many agents such as PMA and actin microfilament disrupters that, in contrast to Mn2+, induce adhesiveness of LFA-1 but not detectable high affinity binding to ICAM-1 (Dransfield et al., 1992). Because of the perceived need to explain this disparity, it was generally accepted prima facie that clusters would be preformed. Our analysis of both macro- and microclustering demonstrate that, in the absence of either a multivalent ligand on a substrate or homotypic cell aggregation, PMA, cytochalasin D, latrunculin A, and chemokine treatments do not alter integrin distribution patterns despite their ability to promote significant adhesion (Fig. 9). These agents failed to increase FRET between neighboring αL-mCFP/β2 and αL-mYFP/β2 molecules or between neighboring αL/β2-mCFP and αL/β2-mYFP molecules. This agrees with our previous observation that stimulation with chemokines, Mn2+ plus sICAM-1, and PMA induced separation between the cytoplasmic domains of the αL and β2 subunits as shown by a decrease in FRET between αL-mCFP/β2-mYFP in individual LFA-1 heterodimers (Kim et al., 2003). Previous experiments designed to assess integrin macroclustering were often performed under conditions that promote robust homotypic cell aggregation; no effort was made to prevent homotypic adhesion, the effect of homotypic adhesion or a preincubation period in monodisperse cell suspension on cluster formation was not tested, and somtimes it was emphasized that after activation cells were fixed immediately before preparation for microscopy.

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