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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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Cytoskeleton disruption leads to accumulation of LFA-1 to the zone of ICAM-1 substrate contact. (A and B) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (A) and without (B) initial centrifugation, to coverslips coated with ICAM-1 or BSA in the absence (Control) or presence of 1 mM Mn2+, 1 μM PMA, or 1 μM cytochalasin D as indicated for 30 min at 37°C. Cells were then fixed and subjected to IRM (top left panels) and DIC (top right panels). The zone including all IRM contacts for each cell was outlined and the area calculated by OpenLab software (bottom panels). Each symbol represents one cell. Bar = mean. *, P < 0.01 vs. Mn2+-treated cells. (C) Cells were prepared as in A and then were additionally subjected to staining with mAbs CBR LFA-1/7-Cy3 to β2 and TS2/4-Cy3 to αL followed by anti–mouse IgG-Cy3 to maximize the fluorescent signal. Samples were then analyzed by serial sectioning (0.5 μm Z-step) confocal microscopy. Images represent either the top view of the three-dimensional image reconstruction for “All” of the sections, or individual sections taken from the “Middle” or “Bottom” (at the ICAM-1 substrate contact interface) planes. Fluorescence intensity of LFA-1 staining in the “Bottom” section was plotted in the three-dimensional histogram of “LFA-1 density”. The LFA-1 accumulation index (AI) was calculated as (total number of pixels in the contact area × mean intensity of those pixels)/(106). AI = mean ± SEM for nine cells. *, P < 0.01 vs. Mn2+-treated cells.
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fig6: Cytoskeleton disruption leads to accumulation of LFA-1 to the zone of ICAM-1 substrate contact. (A and B) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (A) and without (B) initial centrifugation, to coverslips coated with ICAM-1 or BSA in the absence (Control) or presence of 1 mM Mn2+, 1 μM PMA, or 1 μM cytochalasin D as indicated for 30 min at 37°C. Cells were then fixed and subjected to IRM (top left panels) and DIC (top right panels). The zone including all IRM contacts for each cell was outlined and the area calculated by OpenLab software (bottom panels). Each symbol represents one cell. Bar = mean. *, P < 0.01 vs. Mn2+-treated cells. (C) Cells were prepared as in A and then were additionally subjected to staining with mAbs CBR LFA-1/7-Cy3 to β2 and TS2/4-Cy3 to αL followed by anti–mouse IgG-Cy3 to maximize the fluorescent signal. Samples were then analyzed by serial sectioning (0.5 μm Z-step) confocal microscopy. Images represent either the top view of the three-dimensional image reconstruction for “All” of the sections, or individual sections taken from the “Middle” or “Bottom” (at the ICAM-1 substrate contact interface) planes. Fluorescence intensity of LFA-1 staining in the “Bottom” section was plotted in the three-dimensional histogram of “LFA-1 density”. The LFA-1 accumulation index (AI) was calculated as (total number of pixels in the contact area × mean intensity of those pixels)/(106). AI = mean ± SEM for nine cells. *, P < 0.01 vs. Mn2+-treated cells.

Mentions: Based on these results and the finding above that binding of multivalent, soluble ICAM-1 in the presence of either Mn2+ or SDF-1 can drive microcluster formation, we hypothesized that agents that increase diffusivity regulate adhesion primarily by enhancing accumulation of LFA-1 into the zone of contact with ICAM-1 substrates. Examination of the contact zone itself by interference reflection microscopy (IRM) revealed that cytochalasin D–treated cells formed markedly larger contacts (P < 0.01) on ICAM-1 substrates than Mn2+-treated cells (Fig. 6, A and B). PMA-treated cells formed contact zones of intermediate size that were also significantly (P < 0.01) larger than those of Mn2+-treated cells. Furthermore, control cells that received no pretreatment failed to form significant contacts on ICAM-1 substrates (Fig. 6, A and B). Application of force by centrifugation did not alter the pattern of the contact zone or increase the overall contact area, suggesting that differences in contact zones were not a consequence of differential cell deformability. Fluorescence confocal microscopy revealed that the distribution of LFA-1 was not detectably different among Mn2+-, PMA-, cytochalasin D–, and latrunculin A–treated cells at planes above the cell–substrate contact interface (Fig. 6 C, middle). However, at the plane of contact, significantly (P < 0.01) greater total accumulation of LFA-1 was observed with cytochalasin D– and latrunculin A–treated cells than with Mn2+-treated cells (Fig. 6 C, bottom). Compared with Mn2+-treated cells, cytochalasin D– and latrunculin A–treated cells accumulated 5.7- and 7.2-fold more LFA-1 in the substrate contact zone, respectively, whereas PMA-treated cells exhibited a 1.5-fold increase (Fig. 6 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)

Cytoskeleton disruption leads to accumulation of LFA-1 to the zone of ICAM-1 substrate contact. (A and B) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (A) and without (B) initial centrifugation, to coverslips coated with ICAM-1 or BSA in the absence (Control) or presence of 1 mM Mn2+, 1 μM PMA, or 1 μM cytochalasin D as indicated for 30 min at 37°C. Cells were then fixed and subjected to IRM (top left panels) and DIC (top right panels). The zone including all IRM contacts for each cell was outlined and the area calculated by OpenLab software (bottom panels). Each symbol represents one cell. Bar = mean. *, P < 0.01 vs. Mn2+-treated cells. (C) Cells were prepared as in A and then were additionally subjected to staining with mAbs CBR LFA-1/7-Cy3 to β2 and TS2/4-Cy3 to αL followed by anti–mouse IgG-Cy3 to maximize the fluorescent signal. Samples were then analyzed by serial sectioning (0.5 μm Z-step) confocal microscopy. Images represent either the top view of the three-dimensional image reconstruction for “All” of the sections, or individual sections taken from the “Middle” or “Bottom” (at the ICAM-1 substrate contact interface) planes. Fluorescence intensity of LFA-1 staining in the “Bottom” section was plotted in the three-dimensional histogram of “LFA-1 density”. The LFA-1 accumulation index (AI) was calculated as (total number of pixels in the contact area × mean intensity of those pixels)/(106). AI = mean ± SEM for nine cells. *, P < 0.01 vs. Mn2+-treated cells.
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fig6: Cytoskeleton disruption leads to accumulation of LFA-1 to the zone of ICAM-1 substrate contact. (A and B) Stable K562 cell transfectants expressing wild-type αLβ2 were allowed to adhere, with (A) and without (B) initial centrifugation, to coverslips coated with ICAM-1 or BSA in the absence (Control) or presence of 1 mM Mn2+, 1 μM PMA, or 1 μM cytochalasin D as indicated for 30 min at 37°C. Cells were then fixed and subjected to IRM (top left panels) and DIC (top right panels). The zone including all IRM contacts for each cell was outlined and the area calculated by OpenLab software (bottom panels). Each symbol represents one cell. Bar = mean. *, P < 0.01 vs. Mn2+-treated cells. (C) Cells were prepared as in A and then were additionally subjected to staining with mAbs CBR LFA-1/7-Cy3 to β2 and TS2/4-Cy3 to αL followed by anti–mouse IgG-Cy3 to maximize the fluorescent signal. Samples were then analyzed by serial sectioning (0.5 μm Z-step) confocal microscopy. Images represent either the top view of the three-dimensional image reconstruction for “All” of the sections, or individual sections taken from the “Middle” or “Bottom” (at the ICAM-1 substrate contact interface) planes. Fluorescence intensity of LFA-1 staining in the “Bottom” section was plotted in the three-dimensional histogram of “LFA-1 density”. The LFA-1 accumulation index (AI) was calculated as (total number of pixels in the contact area × mean intensity of those pixels)/(106). AI = mean ± SEM for nine cells. *, P < 0.01 vs. Mn2+-treated cells.
Mentions: Based on these results and the finding above that binding of multivalent, soluble ICAM-1 in the presence of either Mn2+ or SDF-1 can drive microcluster formation, we hypothesized that agents that increase diffusivity regulate adhesion primarily by enhancing accumulation of LFA-1 into the zone of contact with ICAM-1 substrates. Examination of the contact zone itself by interference reflection microscopy (IRM) revealed that cytochalasin D–treated cells formed markedly larger contacts (P < 0.01) on ICAM-1 substrates than Mn2+-treated cells (Fig. 6, A and B). PMA-treated cells formed contact zones of intermediate size that were also significantly (P < 0.01) larger than those of Mn2+-treated cells. Furthermore, control cells that received no pretreatment failed to form significant contacts on ICAM-1 substrates (Fig. 6, A and B). Application of force by centrifugation did not alter the pattern of the contact zone or increase the overall contact area, suggesting that differences in contact zones were not a consequence of differential cell deformability. Fluorescence confocal microscopy revealed that the distribution of LFA-1 was not detectably different among Mn2+-, PMA-, cytochalasin D–, and latrunculin A–treated cells at planes above the cell–substrate contact interface (Fig. 6 C, middle). However, at the plane of contact, significantly (P < 0.01) greater total accumulation of LFA-1 was observed with cytochalasin D– and latrunculin A–treated cells than with Mn2+-treated cells (Fig. 6 C, bottom). Compared with Mn2+-treated cells, cytochalasin D– and latrunculin A–treated cells accumulated 5.7- and 7.2-fold more LFA-1 in the substrate contact zone, respectively, whereas PMA-treated cells exhibited a 1.5-fold increase (Fig. 6 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.

Show MeSH
Related in: MedlinePlus