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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: 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.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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Analysis of α4 integrin diffusivity in α4-transfected CHO cells.  40-nm gold particles coated with nonperturbing, anti-α4 mAb (B5G10)  were bound to the surface of α4-transfected CHO cells and tracked in the  plane of the membrane at 37°C, as described in Materials and Methods.  Representative tracks (x versus y; μm) are shown for particles on CHO– α4wt (A) and CHO–X4C0 (B). All tracks were rotated to orient cell with  leading edge facing left. Also, x and y coordinates with respect to time are  shown for CHO–α4wt (C ) and CHO–X4C0 (D) cells (x coordinates, fine  line, ⊥; y coordinates, bold line, ∥). (E) Two-dimensional diffusivity (D;  μm2/s) determined from a plot of MSD versus time of particles tracked  on CHO–α4wt (n = 6) and CHO–X4C0 (n = 9) transfectants (P <0.01).  Data are represented as mean deviation ± SD. No binding of anti-α4-coated particles was detected on mock-transfected CHO cells.
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Figure 6: Analysis of α4 integrin diffusivity in α4-transfected CHO cells. 40-nm gold particles coated with nonperturbing, anti-α4 mAb (B5G10) were bound to the surface of α4-transfected CHO cells and tracked in the plane of the membrane at 37°C, as described in Materials and Methods. Representative tracks (x versus y; μm) are shown for particles on CHO– α4wt (A) and CHO–X4C0 (B). All tracks were rotated to orient cell with leading edge facing left. Also, x and y coordinates with respect to time are shown for CHO–α4wt (C ) and CHO–X4C0 (D) cells (x coordinates, fine line, ⊥; y coordinates, bold line, ∥). (E) Two-dimensional diffusivity (D; μm2/s) determined from a plot of MSD versus time of particles tracked on CHO–α4wt (n = 6) and CHO–X4C0 (n = 9) transfectants (P <0.01). Data are represented as mean deviation ± SD. No binding of anti-α4-coated particles was detected on mock-transfected CHO cells.

Mentions: As illustrated in Fig. 6, A and C, gold particles bound to the lamella of CHO–α4wt cells diffused freely with a mean diffusion coefficient of 0.03 μm2/s (Fig. 6 E), consistent with the diffusion rate observed for other β1 integrins (22), as well as other cell surface glycoproteins (30). However, truncation of the α4 cytoplasmic domain resulted in a significant decrease in the α4β1 diffusion rate (P <0.01). Particles bound to CHO–X4C0 cells exhibited reduced lateral mobility (Fig. 6, B and D), with a diffusion coefficient that was sixfold lower (0.005 μm2/s) than wild-type α4β1. No binding of gold particles was detected on mock-transfected CHO cells, demonstrating that the binding is α4β1 specific (data not shown).


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)

Analysis of α4 integrin diffusivity in α4-transfected CHO cells.  40-nm gold particles coated with nonperturbing, anti-α4 mAb (B5G10)  were bound to the surface of α4-transfected CHO cells and tracked in the  plane of the membrane at 37°C, as described in Materials and Methods.  Representative tracks (x versus y; μm) are shown for particles on CHO– α4wt (A) and CHO–X4C0 (B). All tracks were rotated to orient cell with  leading edge facing left. Also, x and y coordinates with respect to time are  shown for CHO–α4wt (C ) and CHO–X4C0 (D) cells (x coordinates, fine  line, ⊥; y coordinates, bold line, ∥). (E) Two-dimensional diffusivity (D;  μm2/s) determined from a plot of MSD versus time of particles tracked  on CHO–α4wt (n = 6) and CHO–X4C0 (n = 9) transfectants (P <0.01).  Data are represented as mean deviation ± SD. No binding of anti-α4-coated particles was detected on mock-transfected CHO cells.
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Figure 6: Analysis of α4 integrin diffusivity in α4-transfected CHO cells. 40-nm gold particles coated with nonperturbing, anti-α4 mAb (B5G10) were bound to the surface of α4-transfected CHO cells and tracked in the plane of the membrane at 37°C, as described in Materials and Methods. Representative tracks (x versus y; μm) are shown for particles on CHO– α4wt (A) and CHO–X4C0 (B). All tracks were rotated to orient cell with leading edge facing left. Also, x and y coordinates with respect to time are shown for CHO–α4wt (C ) and CHO–X4C0 (D) cells (x coordinates, fine line, ⊥; y coordinates, bold line, ∥). (E) Two-dimensional diffusivity (D; μm2/s) determined from a plot of MSD versus time of particles tracked on CHO–α4wt (n = 6) and CHO–X4C0 (n = 9) transfectants (P <0.01). Data are represented as mean deviation ± SD. No binding of anti-α4-coated particles was detected on mock-transfected CHO cells.
Mentions: As illustrated in Fig. 6, A and C, gold particles bound to the lamella of CHO–α4wt cells diffused freely with a mean diffusion coefficient of 0.03 μm2/s (Fig. 6 E), consistent with the diffusion rate observed for other β1 integrins (22), as well as other cell surface glycoproteins (30). However, truncation of the α4 cytoplasmic domain resulted in a significant decrease in the α4β1 diffusion rate (P <0.01). Particles bound to CHO–X4C0 cells exhibited reduced lateral mobility (Fig. 6, B and D), with a diffusion coefficient that was sixfold lower (0.005 μm2/s) than wild-type α4β1. No binding of gold particles was detected on mock-transfected CHO cells, demonstrating that the binding is α4β1 specific (data not shown).

Bottom Line: 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.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