Isolation of Integrin‐Based Adhesion Complexes

Matthew C. Jones1, Jonathan D. Humphries1, Adam Byron1, Angélique Millon‐Frémillon1, Joseph Robertson1, Nikki R. Paul1, Daniel H. J. Ng1, Janet A. Askari1, Martin J. Humphries1

1 All authors made equal contributions to method development or protocol preparation
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 9.8
DOI:  10.1002/0471143030.cb0908s66
Online Posting Date:  March, 2015
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Abstract

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter‐receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin‐associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin‐associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. © 2015 by John Wiley & Sons, Inc.

Keywords: integrins; cell adhesion; adhesion complexes; extracellular matrix; affinity purification; ventral membranes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Microbead‐Based Isolation of Integrin Adhesion Complexes for Proteomic Analysis
  • Basic Protocol 2: Isolation of Integrin‐Associated Adhesion Complexes from Fibroblasts Attached to a 2‐D Fibronectin Substrate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microbead‐Based Isolation of Integrin Adhesion Complexes for Proteomic Analysis

  Materials
  • Cells (as appropriate; K562 cells used as example)
  • Complete, FBS‐containing cell culture medium (as appropriate)
  • Dynabeads M‐450 Tosylactivated (Dynal Biotech, cat no. 140.13; available from Thermo Fisher Scientific) supplied at 4 × 108 beads/ml in distilled H 2O
  • 1 mg/ml bovine fibronectin (F1141; Sigma‐Aldrich) or other integrin‐specific or control ligands or antibodies
  • 0.1 M sodium phosphate buffer, pH 7.4 to 8 ( )
  • 1% (w/v) bovine serum albumin (BSA; Sigma‐Aldrich; cat. no. A7638) in 0.1 M sodium phosphate buffer, pH 7.4 to 8
  • 0.1% (w/v) bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A7638) in PBS
  • 0.1% (w/v) bovine serum albumin (BSA) in 0.2 M Tris·Cl, pH 8.5 (see for the Tris buffer)
  • Dulbecco's modified Eagle's medium containing 25 mM HEPES (DMEM‐HEPES; Sigma‐Aldrich, cat. no. D6171; also available from Thermo Fisher Scientific)
  • 0.2% (w/v) bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A7638) in DMEM‐HEPES
  • PBS: calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; )
  • Trypsin‐EDTA solution (available from various molecular biology suppliers)
  • Manganese chloride (Sigma‐Aldrich)
  • DTBP crosslinker (Thermo Fisher, cat. no. 20665)
  • 1× RIPA buffer (for microbead assay; see recipe)
  • 5× reducing sample buffer (see recipe)
  • Cytoskeletal stabilizing buffers (prepare as described in Reagents and Solutions):
    • CSK (see recipe)
    • CSK with Tris (see recipe)
    • CSK+ (see recipe)
  • 0.2% (w/v) bovine serum albumin in DMEM‐HEPES
  • 0.1% (w/v) crystal violet
  • 10% (v/v) acetic acid
  • Temperature‐controlled shaking incubator for bead coating and recovery of adhesion complexes (Thermomixer compact; Eppendorf)
  • Magnetic particle separator (MPC; Dynal, Thermo Fisher Scientific): MPC‐S for 1.5‐ml microcentrifuge tubes and MPC‐6 for 14‐ml conical tubes
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Refrigerated centrifuge
  • Platform shaker
  • Microscope slides and coverslips
  • Light microscope
  • 96‐well plates (Corning Costar, cat. no. 3596)
  • 96‐well plate reader spectrophotometer
  • Bioruptor UCD‐200 (Diagenode) or Sonicator VibraCell VCX 500, (Sonics & Materials)
  • Additional reagents and equipment for basic cell culture techniques including trypsinization and counting cells (unit )

Basic Protocol 2: Isolation of Integrin‐Associated Adhesion Complexes from Fibroblasts Attached to a 2‐D Fibronectin Substrate

  Materials
  • 10 μg/ml bovine fibronectin (F1141; Sigma‐Aldrich) in Dulbecco's phosphate‐buffered saline (DPBS; with Ca2+ and Mg2+; ) prepared fresh from stock solutions immediately before use
  • 10 μg/ml apotransferrin (Sigma‐Aldrich, cat. no. T5391) or 10 μg/ml poly‐D‐lysine (M r 70,000 to 150,000; Sigma‐Aldrich, cat. no. P6407) in Dulbecco's phosphate‐buffered saline (DPBS, with Ca2+ and Mg2+; )
  • PBS: Dulbecco's phosphate‐buffered saline without Ca2+ or Mg2+ (CMF‐DPBS; )
  • Heat‐denatured BSA solution (see recipe)
  • Human foreskin fibroblast (HFF) cells growing in 10‐cm plates at 70% to 80% confluence
  • Dulbecco's Modified Eagle Medium (Sigma‐Aldrich, cat. no. D5796), serum‐free
  • DTBP crosslinker (Thermo Fisher 20665)
  • Dulbecco's modified Eagle's medium containing 25 mM HEPES (DMEM‐HEPES; Invitrogen or Sigma‐Aldrich D6171)
  • 1 M Tris·Cl, pH 8 ( )
  • Modified RIPA buffer (see recipe)
  • Adhesion recovery solution (see recipe)
  • Acetone, −20°C
  • 5× reducing sample buffer (see recipe)
  • 100 × 20–mm (10‐cm) sterile tissue culture plates (Corning Costar, cat. no. 430167)
  • Cell scrapers (Greiner Bio‐One GmbH, cat. no. 541 070)
  • Light microscope
  • Water tap with 8‐mm‐diameter plastic tubing attached
  • Refrigerated benchtop microcentrifuge capable of holding 1.6‐ml tubes and centrifugation at 22,000 × g
  • Additional reagents and equipment for basic cell culture techniques including trypsinization and counting cells (unit )
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Figures

Videos

Literature Cited

Literature Cited
   Beacham, D.A. , Amatangelo, M.D. , and Cukierman, E. 2006. Preparation of extracellular matrices produced by cultured and primary fibroblasts. Curr. Protoc. Cell Biol. 33:10.9.1‐10.9.21.
   Byron, A. , Humphries, J.D. , Bass, M.D. , Knight, D. , and Humphries, M.J. 2011. Proteomic analysis of integrin adhesion complexes. Sci. Signal. 4:pt2.
   Byron, A. , Humphries, J.D. , Craig, S.E. , Knight, D. , and Humphries, M.J. 2012. Proteomic analysis of α4β1 integrin adhesion complexes reveals α‐subunit‐dependent protein recruitment. Proteomics 12:2104‐2114.
   Humphries, M.J. 1998. Cell‐substrate adhesion assays. Cur. Protoc. Cell Biol. 00:9.1.1‐9.1.11.
   Humphries, J.D. , Byron A. , Bass M.D. , Craig S.E. , Pinney J.W. , Knight D. , and Humphries, M.J. 2009. Proteomic analysis of integrin‐associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Sci. Signal. 2:ra51.
   Kuo, J‐C. , Han, X. , Hsiao, C‐T. , Yates, III, J.R. , and Waterman, C.M. 2011. Analysis of the myosin‐II‐responsive focal adhesion proteome reveals a role for β‐Pix in negative regulation of focal adhesion maturation. Nat. Cell Biol. 13:383‐393.
   Schiller, H.B. , Friedel, C.C. , Boulegue, C. , and Fässler, R. 2011. Quantitative proteomics of the integrin adhesome show a myosin II‐dependent recruitment of LIM domain proteins. EMBO Rep. 12:259‐266.
   Schiller, H.B. , Hermann, M.R. , Polleux, J. , Vignaud, T. , Zanivan, S. , Friedel, C.C. , Sun, Z. , Raducanu, A. , Gottschalk, K.E. , Théry, M. , Mann, M. , and Fässler, R. 2013. β1‐ and αv‐class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin‐based microenvironments. Nat. Cell Biol. 15:625‐636.
   Wehrle‐Haller, B. 2012. Structure and function of focal adhesions. Curr. Opin. Cell Biol. 24:116‐124.
   Winograd‐Katz, S.E. , Fässler, R. , Geiger, B. , and Legate, K.R. 2014. The integrin adhesome: From genes and proteins to human disease. Nat. Rev. Mol. Cell Biol. 15:273‐288.
   Wolfenson, H. , Lavelin, I. , and Geiger, B. 2013. Dynamic regulation of the structure and functions of integrin adhesions. Dev. Cell. 24:447‐458.
   Zaidel‐Bar, R. , Itzkovitz, S. , Ma'ayan, A. , Iyengar, R. , and Geiger, B. 2007. Functional atlas of the integrin adhesome. Nat. Cell Biol. 9:858‐867.
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