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First published online 22 December 2004
doi: 10.1242/jcs.01623

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A specific ₅ß₁-integrin conformation promotes directional integrin translocation and fibronectin matrix formation

Katherine Clark¹, Roumen Pankov¹, Mark A. Travis^2,*, Janet A. Askari², A. Paul Mould², Susan E. Craig², Peter Newham^2,, Kenneth M. Yamada¹ and Martin J. Humphries^2,

¹ Craniofacial Developmental Biology and Regeneration Branch, NIDCR, NIH, Bethesda, MA 20892, USA
² Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (13K): [in a new window]   Fig. 1. K562-cell adhesion to fibronectin is promoted by SNAKA51 and other stimulatory anti-ß1-integrin antibodies. K562 cells were allowed to attach to a fibronectin-coated surface (2 µg/ml) in the presence of the indicated anti-integrin antibodies (10 µg/ml), PMA (100 nM) or no antibody (control). Unattached cells were removed, and remaining cells were fixed and stained with Crystal Violet. Cell attachment was quantified by absorbance measured at 600 nm.

View larger version (19K): [in a new window]   Fig. 2. SNAKA51 promotes ligand-binding to 5ß1 integrin, and SNAKA51 binding to 5ß1 integrin is increased in the presence of ligand. Using a solid-phase ligand-binding assay, the binding of biotinylated FnIII (6-10) (0.1 µg/ml) to 5ß1 integrin was measured. (a) Binding of FnIII (6-10) to directly coated placental 5ß1 integrin and to anti-Fc, captured recombinant integrin in the presence and absence of SNAKA51, 12G10, or K20. Statistical analysis was performed using a 2-tailed t-test in comparison to the control without antibody. * P<0.005, **P<0.0005. (b) Dose-dependent binding of biotinylated SNAKA51 to K20 tethered placental 5ß1 integrin in the presence or absence of FnIII (6-10) (20 µg/ml).

View larger version (53K): [in a new window]   Fig. 3. SNAKA51 only colocalizes with fibronectin fibers and primed ß1 integrin in fibrillar adhesions. Human fibroblasts were fixed and stained using indirect immunofluorescence. (a) 5 integrin [SNAKA51 (left), SNAKA52 (middle), mAb11 (right), all green], fibronectin (blue) and V integrin (red) as a focal adhesion marker (L230). (b) ß1 integrin in a primed conformation (9EG7) (red) and 5 integrin (SNAKA51, green) (top). Total ß1 integrin (mAb11 red) and 5 integrin (SNAKA51, green) (middle). ß1 integrin in a primed conformation (9EG7, red) and total ß1 integrin (mAb11, green) (bottom). Bar 20 µm, inset bar 5 µm.

View larger version (20K): [in a new window]   Fig. 4. The SNAKA51 epitope maps to the calf domains of the 5 subunit. (a) The extracellular domains of the 5- and ß1-integrin subunits indicate the position of truncations for the various recombinant constructs used in this study. (b) The SNAKA51 binding site on the 5 integrin subunit was mapped using a sandwich ELISA of Fc-tagged truncated or individual domains of recombinant integrin. The Fc-tagged protein was specifically captured with anti-Fc antibodies; binding of SNAKA51 to the integrin was measured with enzyme-conjugated anti-mouse secondary antibody.

View larger version (16K): [in a new window]   Fig. 5. Induction of priming by SNAKA51 depends on the ß1-integrin leg and modulates ß1-integrin A-domain ligand-binding ability. Binding of biotinylated FnIII (6-10) to either wild-type or ADMIDAS mutant (D138A) recombinant 5ß1 integrin, containing either a full-length or truncated (455) ß1 subunit, was measured in the presence or absence of anti-integrin antibodies.

View larger version (61K): [in a new window]   Fig. 6. Clustering of SNAKA51-bound 5 integrin induces integrin translocation out of focal adhesions and across the cell surface. Fibroblasts were plated on glass coverslips and incubated overnight in fibronectin-depleted medium with cycloheximide. Cells were labeled with antibody for 20 minutes, using (a) SNAKA51, (b) SNAKA52 or (c) mAb11. Unbound antibody was rinsed off and the cells were either fixed (no chasing) or the cell-bound antibody was clustered with goat anti-mouse or anti-rat IgG and incubated for a further 30 minutes ('30' chasing) before fixation. For unclustered 5 integrin, cells were stained with anti-mouse or anti-rat IgG (green). For clustered 5 integrin, cells were stained with anti-goat IgG (green). In addition, all samples were stained with anti-V antibody L230 (red) as a focal adhesion marker. Bar 20 µm.

View larger version (20K): [in a new window]   Fig. 7. Human salivary gland cells (HSG) were treated (a) overnight with a range of concentrations of biotinylated fibronectin, or (b) with biotinylated fibronectin (10 mg/ml) for different times. (c) Cells were treated with biotinylated fibronectin (10 mg/ml) together with anti-integrin antibodies (25 mg/ml), or (d) with a range of concentrations of SNAKA51 antibody. The cells were extracted with deoxycholate buffer, and the insoluble matrix fraction was collected and analyzed in a western blot. The upper part of each panel indicates biotinylated fibronectin incorporation into the insoluble matrix fraction. The lower part of each panel indicates cytokeratin as internal loading controls for the HSG cells.

View larger version (20K): [in a new window]   Fig. 8. Model of transitions of 5ß1 integrin for fibrillar-adhesion-formation and fibronectin fibrillogenesis. (a) Predicted domain structure of 5ß1 integrin. The green box (calf domains) indicates the region containing the SNAKA51 epitope. Blue circles represent cations bound to the MIDAS and ADMIDAS sites in the ß-integrin A-domain. (b) (1) Inactive integrin is diffusely located on the cell surface. (2) 5ß1 located in focal adhesions expresses epitopes reporting a primed ß1 conformation (e.g. 9EG7). These integrins may or may not be fully bound by ligand. (3) Integrin located at the distal edge of focal adhesions has additional SNAKA51 epitope expression. Clustering of this integrin promotes translocation. (4) Ligated and clustered integrin translocates out of focal adhesions along the actin cytoskeleton, stretching extracellular fibronectin fibrils and driving fibrillogenesis.