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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 enhances cell adhesion but does not promote soluble ligand binding or conformational change. (A and B) K562 transfectants expressing wild-type αLβ2 were preincubated with Mn2+, PMA, cytochalasin D, or latrunculin A in the absence or presence of inhibitory αL mAb TS1/22 (10 μg/ml) and allowed to bind ICAM-1 immobilized on V-bottom (A) or flat-bottom (B) plates, as described in Materials and methods. Data represent mean ± SEM of all measurements from three independent experiments in duplicate (A) or three independent experiments in triplicate (B). (C and D) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (C) and without (D) an initial centrifugation, to coverslips coated with ICAM-1 in the absence (Control) or presence of 1 mM Mn2+ or 1 μM cytochalasin D for 30 min at 37°C under static conditions. Coverslips were transferred to a laminar flow chamber and cells were then detached by a shear regimen of 30 s each of 0, 2, 4, 8, 16, and 32 dyn/cm2. The number of cells remaining after each interval were counted. Values are mean ± SEM for three separate experiments. (E) Binding of soluble, multimeric ICAM-1-Fcα chimera/anti-IgA-FITC complex was conducted in the presence of Mn2+, PMA, cytochalasin D, or latrunculin A at 37°C and measured by flow cytometry. Data show mean ± SEM of three experiments, each in triplicate. (F) Binding of conformation-sensitive antibodies KIM127 and m24 in the absence or presence of Mn2+, PMA, cytochalasin D, or latrunculin A was measured by immunofluorescence flow cytometry. A representative of three separate experiments is shown.
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fig3: Disruption of cytoskeletal constraints enhances cell adhesion but does not promote soluble ligand binding or conformational change. (A and B) K562 transfectants expressing wild-type αLβ2 were preincubated with Mn2+, PMA, cytochalasin D, or latrunculin A in the absence or presence of inhibitory αL mAb TS1/22 (10 μg/ml) and allowed to bind ICAM-1 immobilized on V-bottom (A) or flat-bottom (B) plates, as described in Materials and methods. Data represent mean ± SEM of all measurements from three independent experiments in duplicate (A) or three independent experiments in triplicate (B). (C and D) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (C) and without (D) an initial centrifugation, to coverslips coated with ICAM-1 in the absence (Control) or presence of 1 mM Mn2+ or 1 μM cytochalasin D for 30 min at 37°C under static conditions. Coverslips were transferred to a laminar flow chamber and cells were then detached by a shear regimen of 30 s each of 0, 2, 4, 8, 16, and 32 dyn/cm2. The number of cells remaining after each interval were counted. Values are mean ± SEM for three separate experiments. (E) Binding of soluble, multimeric ICAM-1-Fcα chimera/anti-IgA-FITC complex was conducted in the presence of Mn2+, PMA, cytochalasin D, or latrunculin A at 37°C and measured by flow cytometry. Data show mean ± SEM of three experiments, each in triplicate. (F) Binding of conformation-sensitive antibodies KIM127 and m24 in the absence or presence of Mn2+, PMA, cytochalasin D, or latrunculin A was measured by immunofluorescence flow cytometry. A representative of three separate experiments is shown.

Mentions: Valency-based modes of integrin regulation are known to act, at least in part, through increasing integrin cell surface diffusivity (Kucik et al., 1996; Zhou et al., 2001). Cytochalasin D and latrunculin A have been used previously to dissect integrin regulatory mechanisms, and are thought to act by selectively increasing integrin diffusivity without altering conformation (Kucik et al., 1996; van Kooyk and Figdor, 2000; Zhou et al., 2001). PMA has been reported both to modulate affinity and to increase diffusivity (Lollo et al., 1993; Kucik et al., 1996; Zhou et al., 2001). Consistent with previous reports (Kucik et al., 1996; van Kooyk and Figdor, 2000; Zhou et al., 2001), cell pretreatment with 1 μM cytochalasin D or 1 μM latrunculin A caused partial disruption of actin filaments in K562 cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404160/DC1) and significantly increased LFA-1 lateral mobility (Fig. S4) as measured by Alexa 488–phalloidin staining and FRAP, respectively. The same concentrations of cytochalasin D and latrunculin A induced significant adhesion in both V-bottom (Fig. 3 A) and conventional flat-bottom well (Fig. 3 B) assays, which was comparable to that induced by 1 mM Mn2+. Substantial adhesion promoted by 1 μM PMA was readily detected by the V-bottom assay (Fig. 3 A), but not by the less sensitive flat-bottom assay (Fig. 3 B). In the flat-bottom assay cells are washed with high shear, whereas in the V-bottom assay nonadherent cells are separated by centrifugation and there is no washing. Adhesion stimulated by cytochalasin D was further analyzed under physiologic shear conditions in a parallel wall flow chamber (Fig. 3, C and D). Cytochalasin D promoted significant amounts of highly shear-resistant cellular adhesions either with (Fig. 3 C) or without (Fig. 3 D) brief precentrifugation to promote initial contacts, and was only somewhat less effective than Mn2+.


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 enhances cell adhesion but does not promote soluble ligand binding or conformational change. (A and B) K562 transfectants expressing wild-type αLβ2 were preincubated with Mn2+, PMA, cytochalasin D, or latrunculin A in the absence or presence of inhibitory αL mAb TS1/22 (10 μg/ml) and allowed to bind ICAM-1 immobilized on V-bottom (A) or flat-bottom (B) plates, as described in Materials and methods. Data represent mean ± SEM of all measurements from three independent experiments in duplicate (A) or three independent experiments in triplicate (B). (C and D) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (C) and without (D) an initial centrifugation, to coverslips coated with ICAM-1 in the absence (Control) or presence of 1 mM Mn2+ or 1 μM cytochalasin D for 30 min at 37°C under static conditions. Coverslips were transferred to a laminar flow chamber and cells were then detached by a shear regimen of 30 s each of 0, 2, 4, 8, 16, and 32 dyn/cm2. The number of cells remaining after each interval were counted. Values are mean ± SEM for three separate experiments. (E) Binding of soluble, multimeric ICAM-1-Fcα chimera/anti-IgA-FITC complex was conducted in the presence of Mn2+, PMA, cytochalasin D, or latrunculin A at 37°C and measured by flow cytometry. Data show mean ± SEM of three experiments, each in triplicate. (F) Binding of conformation-sensitive antibodies KIM127 and m24 in the absence or presence of Mn2+, PMA, cytochalasin D, or latrunculin A was measured by immunofluorescence flow cytometry. A representative of three separate experiments is shown.
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fig3: Disruption of cytoskeletal constraints enhances cell adhesion but does not promote soluble ligand binding or conformational change. (A and B) K562 transfectants expressing wild-type αLβ2 were preincubated with Mn2+, PMA, cytochalasin D, or latrunculin A in the absence or presence of inhibitory αL mAb TS1/22 (10 μg/ml) and allowed to bind ICAM-1 immobilized on V-bottom (A) or flat-bottom (B) plates, as described in Materials and methods. Data represent mean ± SEM of all measurements from three independent experiments in duplicate (A) or three independent experiments in triplicate (B). (C and D) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (C) and without (D) an initial centrifugation, to coverslips coated with ICAM-1 in the absence (Control) or presence of 1 mM Mn2+ or 1 μM cytochalasin D for 30 min at 37°C under static conditions. Coverslips were transferred to a laminar flow chamber and cells were then detached by a shear regimen of 30 s each of 0, 2, 4, 8, 16, and 32 dyn/cm2. The number of cells remaining after each interval were counted. Values are mean ± SEM for three separate experiments. (E) Binding of soluble, multimeric ICAM-1-Fcα chimera/anti-IgA-FITC complex was conducted in the presence of Mn2+, PMA, cytochalasin D, or latrunculin A at 37°C and measured by flow cytometry. Data show mean ± SEM of three experiments, each in triplicate. (F) Binding of conformation-sensitive antibodies KIM127 and m24 in the absence or presence of Mn2+, PMA, cytochalasin D, or latrunculin A was measured by immunofluorescence flow cytometry. A representative of three separate experiments is shown.
Mentions: Valency-based modes of integrin regulation are known to act, at least in part, through increasing integrin cell surface diffusivity (Kucik et al., 1996; Zhou et al., 2001). Cytochalasin D and latrunculin A have been used previously to dissect integrin regulatory mechanisms, and are thought to act by selectively increasing integrin diffusivity without altering conformation (Kucik et al., 1996; van Kooyk and Figdor, 2000; Zhou et al., 2001). PMA has been reported both to modulate affinity and to increase diffusivity (Lollo et al., 1993; Kucik et al., 1996; Zhou et al., 2001). Consistent with previous reports (Kucik et al., 1996; van Kooyk and Figdor, 2000; Zhou et al., 2001), cell pretreatment with 1 μM cytochalasin D or 1 μM latrunculin A caused partial disruption of actin filaments in K562 cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404160/DC1) and significantly increased LFA-1 lateral mobility (Fig. S4) as measured by Alexa 488–phalloidin staining and FRAP, respectively. The same concentrations of cytochalasin D and latrunculin A induced significant adhesion in both V-bottom (Fig. 3 A) and conventional flat-bottom well (Fig. 3 B) assays, which was comparable to that induced by 1 mM Mn2+. Substantial adhesion promoted by 1 μM PMA was readily detected by the V-bottom assay (Fig. 3 A), but not by the less sensitive flat-bottom assay (Fig. 3 B). In the flat-bottom assay cells are washed with high shear, whereas in the V-bottom assay nonadherent cells are separated by centrifugation and there is no washing. Adhesion stimulated by cytochalasin D was further analyzed under physiologic shear conditions in a parallel wall flow chamber (Fig. 3, C and D). Cytochalasin D promoted significant amounts of highly shear-resistant cellular adhesions either with (Fig. 3 C) or without (Fig. 3 D) brief precentrifugation to promote initial contacts, and was only somewhat less effective than Mn2+.

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