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First published online 28 February 2006
doi: 10.1242/jcs.02827

Journal of Cell Science 119, 1175-1183 (2006)
Published by The Company of Biologists 2006
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Displacement of the ß cytoplasmic domain recovers focal adhesion formation, cytoskeletal organization and motility in swapped integrin chimeras

Michael A. Partridge*, Frank S. David*,{ddagger} and Eugene E. Marcantonio§

Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA


Figure 1
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  		Fig. 1. Representations of the human {alpha}1 and chicken ß1 collagen IV receptor and the swapped integrin chimera. (A) HA1, or wild-type receptors, express full-length {alpha}1 integrin. (B) SW1 receptors have the extracellular regions swapped onto their opposing transmembrane and cytoplasmic domains (Briesewitz et al., 1995Go). (C) SßX receptors are altered from the SW1 chimera by extension of the ß cytoplasmic domain by 10 residues. (D) Two constructs with 10-residue insertions (bold) were engineered: SßX-ßcyt, a duplication of the first 10 residues of the ß cytoplasmic domain (underlined); and SßX-X10, a random sequence predicted to be {alpha}-helical.

 

Figure 2
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  		Fig. 2. Analysis of FA protein phosphotyrosine levels. Biochemical analysis of FA protein phosphotyrosine levels in cells expressing wild-type human {alpha}1 (HA1) or the swapped integrin chimers (SW1 or SßX). Cells were kept in suspension for 1 hour and allowed to spread on collagen IV (CN) or fibronectin (FN) for 1 hour, and phosphotyrosine levels were compared with the suspension control (S). (A) Phosphorylation of FAK at Y397 and Y576 were detected with site-specific pY antibodies (Biosource). Lysates were used neat (50 µg) for pY397 blots or immunoprecipitated (IP; 500 µg) with anti-FAK mAb (Upstate) for pY576 blots. Membranes were reprobed with anti-FAK (Transduction) or anti-talin (Sigma) mAbs. (B) Total phosphotyrosine levels in immunoprecipitates of paxillin or Cas. Both SW1 and SßX cells mediate normal FAK phosphorylation but are defective in Cas phosphorylation. SßX cells mediate wild-type levels of paxillin phosphorylation.

 

Figure 3
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  		Fig. 3. Morphology of SW1 and SßX cells on collagen IV. Cells were allowed to spread on collagen IV for 1 hour and SW1 (A) and SßX-X10 (B) cells stained for F-actin with Rhodamine-Phalloidin or with antibody to the {alpha}1 integrin extracellular domain to identify the chimeric receptors. Bar, 10 µm.

 

Figure 4
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  		Fig. 4. Localization of FA proteins to the SßX receptor in cells spreading on collagen IV. (A) Cells expressing SßX-ßcyt were allowed to spread on collagen IV and stained for the SßX receptor (anti-{alpha}1 integrin) and with antibodies to either paxillin pY118 or FAK pY397. (B) Cells expressing SßX-ßcyt transiently transfected with either Cas-GFP or GFP-FAK were allowed to spread on fibronectin (FN) or collagen IV (CN) and stained for F-actin with Rhodamine-Phalloidin. Spreading on fibronectin indicates that Cas-GFP was able to localize to FAs generated with wild-type integrins whereas cells transfected with GFP-FAK verify that transiently transfected fluorescently tagged FA protein constructs were able to localize to radial FA-like structures formed by SßX cells on collagen IV. SßX-ßcyt (C) or SW1 (D) cells were transiently transfected with GFP-talin, allowed to spread on collagen IV and stained for the chimeric receptors. Lower panels in C are a higher magnification of the regions outlined in images of the whole cell. Talin, paxillin and FAK, but not Cas, localize to FA-like structures formed with SßX cells on collagen IV.

 

Figure 5
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  		Fig. 5. Formation of stable microtubules in cells expressing HA1, SW1 or SßX integrin. (A,B) SW1 and SßX (SßX-ß-cyt) cells were allowed to spread on collagen IV and stained for vinculin to identify FA structures and Glu-tubulin to reveal stabilized microtubules (Glu-MT). Bar 10 µm. (C) HA1 cells and SßX cells were allowed to spread on collagen IV with or without 1% calf serum (CS) and stained with anti-{alpha}1 (anti-{alpha}1) integrin to identify the HA1 and SßX integrins, and Glu-MT to reveal stabilized microtubules. Only in the presence of serum do SßX, but not SW1, cells form stable microtubules on collagen IV.

 

Figure 6
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  		Fig. 6. Rac and Rho activation by integrins. Activation of the small GTPases Rac and Rho in response to adhesion in parental NIH3T3 cells on fibronectin and cells expressing HA1, SW1 or SßX on collagen IV (CN). Cells were maintained in 1% serum overnight, placed in suspension for 1 hour and plated in 1% serum (Ren et al., 1999Go) on fibronectin or collagen for the times indicated. (A,B) Lysates were incubated with GST-PBD beads for 30 minutes and bound GTP-Rac detected by western blotting. Total cell lysates probed for Rac demonstrated equal amounts of Rac. (C) Graphical representation of the relative increase in GTP-Rac for each of the experiments. Relative GTP-Rac was normalized around levels obtained at 0 minutes adhesion time and adjusted for total Rac loading. (D) SßX cells were plated on fibronectin or collagen IV, lysates were incubated with GST-Rhotekin-RBD and bound GTP-Rho detected by western blotting. Total cell lysates probed for Rho demonstrate equal amounts of protein. Activation of Rac and Rho in response to collagen IV is normal for wild-type {alpha}1-expressing cells but defective for swapped integrin chimeras.

 

Figure 7
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  		Fig. 7. Random migration of cells expressing HA1, SW1 or SßX on collagen IV or fibronectin. Cells were sparsely plated onto glass-bottomed culture dishes coated with fibronectin (FN) or collagen IV (CN) and allowed to spread for 1 hour. Dishes were then placed on a heated microscope stage and images captured every 20 minutes for 10-12 hours. Data are average velocity measured by tracking cell nuclei of at least 10 cells from each of two (HA1 and SW1) or three (SßX) experiments (±s.e.m.). Bars with different shadings are significantly different from each other (Student's t test, P<0.01).

 

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