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Mutational evidence for control of cell adhesion through integrin diffusion/clustering, independent of ligand binding.

Yauch RL, Felsenfeld DP, Kraeft SK, Chen LB, Sheetz MP, Hemler ME - J. Exp. Med. (1997)

Bottom Line: Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique.Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion.Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

ABSTRACT
Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

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Distribution of α4 on multiple K562–α4wt and K562–X4C0 cells. Confocal microscopy was used to examine the cell surface distribution of  α4 upon treatment of K562–α4wt (a and c) and K562–X4C0 (b and d ) transfectants with soluble VCAM (a and b) or with secondary antibodies (c and d ),  as described in Fig. 4. Bar, 10 μm.
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Figure 5: Distribution of α4 on multiple K562–α4wt and K562–X4C0 cells. Confocal microscopy was used to examine the cell surface distribution of α4 upon treatment of K562–α4wt (a and c) and K562–X4C0 (b and d ) transfectants with soluble VCAM (a and b) or with secondary antibodies (c and d ), as described in Fig. 4. Bar, 10 μm.

Mentions: To extend our findings, we also examined α4 clustering induced by anti-α4 mAb, followed by polyclonal secondary antibody (Fig. 4, f–j). As indicated, clustering was again pronounced on K562–α4wt (Fig. 4, i and j) and K562– X4C2 (Fig. 4 h) cells, whereas minimal clustering was observed when the α4 tail was deleted (K562–X4C0 cells; Fig. 4 g) or when no α4 was present (Fig. 4 f  ). Results in Fig. 5, showing 9–15 cells/panel, confirm the single cell results shown in Fig. 4. As indicated, nearly all of the K562– α4wt cells exhibit pronounced clustering, induced either by VCAM (Fig. 5 a) or by antibody (Fig. 5 c). In contrast, the X4C0 mutant was much less clustered (Fig. 5, b and d ), despite being expressed on the cell surface at levels nearly equivalent to α4wt (see Fig. 1).


Mutational evidence for control of cell adhesion through integrin diffusion/clustering, independent of ligand binding.

Yauch RL, Felsenfeld DP, Kraeft SK, Chen LB, Sheetz MP, Hemler ME - J. Exp. Med. (1997)

Distribution of α4 on multiple K562–α4wt and K562–X4C0 cells. Confocal microscopy was used to examine the cell surface distribution of  α4 upon treatment of K562–α4wt (a and c) and K562–X4C0 (b and d ) transfectants with soluble VCAM (a and b) or with secondary antibodies (c and d ),  as described in Fig. 4. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199079&req=5

Figure 5: Distribution of α4 on multiple K562–α4wt and K562–X4C0 cells. Confocal microscopy was used to examine the cell surface distribution of α4 upon treatment of K562–α4wt (a and c) and K562–X4C0 (b and d ) transfectants with soluble VCAM (a and b) or with secondary antibodies (c and d ), as described in Fig. 4. Bar, 10 μm.
Mentions: To extend our findings, we also examined α4 clustering induced by anti-α4 mAb, followed by polyclonal secondary antibody (Fig. 4, f–j). As indicated, clustering was again pronounced on K562–α4wt (Fig. 4, i and j) and K562– X4C2 (Fig. 4 h) cells, whereas minimal clustering was observed when the α4 tail was deleted (K562–X4C0 cells; Fig. 4 g) or when no α4 was present (Fig. 4 f  ). Results in Fig. 5, showing 9–15 cells/panel, confirm the single cell results shown in Fig. 4. As indicated, nearly all of the K562– α4wt cells exhibit pronounced clustering, induced either by VCAM (Fig. 5 a) or by antibody (Fig. 5 c). In contrast, the X4C0 mutant was much less clustered (Fig. 5, b and d ), despite being expressed on the cell surface at levels nearly equivalent to α4wt (see Fig. 1).

Bottom Line: Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique.Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion.Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

ABSTRACT
Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

Show MeSH
Related in: MedlinePlus