Analyzing Integrin‐Dependent Adhesion

A. Paul Mould1

1 University of Manchester, Manchester, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 9.4
DOI:  10.1002/0471143030.cb0904s53
Online Posting Date:  December, 2011
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Abstract

In this unit, methods for the analysis of integrin‐dependent adhesion are described. Two major types of assays are commonly used for this analysis. The first are cell adhesion assays (as described in UNIT 9.1). A key application of this type of assay is to identify which integrin(s) mediate cell‐substrate interactions; a comprehensive list of antibodies suitable for this purpose is detailed. The second are solid‐phase assays in which purified integrins and integrin ligands are used. These assays can be used, e.g., to measure apparent affinities of integrins for different ligands and IC50 values of pharmacological inhibitors. Curr. Protoc. Cell Biol. 53:9.4.1‐9.4.17. © 2011 by John Wiley & Sons, Inc.

Keywords: cell biology; adhesion; extracellular matrix

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analyzing Integrin‐Dependent Adhesion in Cell‐Based Assays
  • Basic Protocol 2: Analyzing Integrin‐Ligand Interactions in Solid‐Phase Assays
  • Support Protocol 1: Integrin Purification
  • Support Protocol 2: Coupling of Antibodies to Sepharose
  • Support Protocol 3: Biotinylation of Integrin Ligands
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analyzing Integrin‐Dependent Adhesion in Cell‐Based Assays

  Materials
  • Adhesion molecule of interest
  • Dulbecco's phosphate‐buffered saline (DPBS; Life Technologies; also see appendix 2A)
  • 10 mg/ml heat‐denatured BSA solution (unit 9.1)
  • Cells of interest
  • DMEM/HEPES: Dulbecco's modified Eagle medium (Life Technologies; appendix 2B) with 25 mM HEPES, gassed with 5% to 10% CO 2
  • Inhibitor: integrin‐specific monoclonal antibody (see Table 9.4.1) or peptide, dissolved in DPBS at appropriate concentration
  • 96‐well tissue culture microtiter plate
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1) and spreading or attachment cell‐substrate adhesion assays (unit 9.1)

Basic Protocol 2: Analyzing Integrin‐Ligand Interactions in Solid‐Phase Assays

  Materials
  • Purified integrin [see protocol 3; several purified and many recombinant integrins are also available commercially (Millipore and R&D Systems, respectively)]
  • Dulbecco's phosphate‐buffered saline (DPBS; Life Technologies; also see appendix 2A)
  • Blocking solution (see recipe)
  • Biotin‐labeled ligand (see protocol 5)
  • Binding buffer (see recipe)
  • ExtrAvidin peroxidase reagent (Sigma‐Aldrich)
  • ABTS reagent (see recipe)
  • 2% (w/v) SDS in water ( appendix 2A)
  • ELISA plate (96‐well EIA/RIA half area plates, Costar)
  • Plastic film (e.g., Nescofilm, Parafilm, Saran wrap)
  • Multichannel pipettor
  • 21‐G hypodermic needle
  • Side‐arm flask
  • Microtiter plate reader

Support Protocol 1: Integrin Purification

  Materials
  • Human placenta (from maternity unit of local hospital; process within a few hours of delivery)
  • Homogenization buffer (see recipe)
  • 1% (w/v) Virkon (Merck) in water
  • Extraction buffer (see recipe), 4°C
  • Sepharose 4B resin (Sigma‐Aldrich)
  • Rat IgG–Sepharose resin (see protocol 4)
  • mAb 13 (anti‐β1)–Sepharose and mAb 16 (anti‐α5)–Sepharose (see protocol 4)
  • Wash buffer (see recipe), 4°C
  • Elution buffer (see recipe), 4°C
  • 1 M Tris⋅Cl, pH 8.2 ( appendix 2A; store up to 6 months at 4°C), 4°C
  • 0.1 M Tris⋅Cl, pH 8.3 ( appendix 2A)/0.1% (w/v) Triton X‐100 (Ultra grade, Sigma‐Aldrich), 4°C (store up to 3 months at 4°C)
  • Phosphate‐buffered saline (PBS; prepare using 10× stock solution from Life Technologies) containing 0.05% (w/v) sodium azide (add from 20% w/v sodium azide stock in H 2O)
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • 5× SDS‐PAGE sample buffer (see recipe)
  • 6% or 7%SDS‐PAGE gel (unit 6.1)
  • Large scissors
  • Blender
  • Beckman J6‐B centrifuge with JA‐10 and JA‐20 rotors (or equivalent refrigerated centrifuge)
  • 500‐ml polycarbonate centrifuge bottles (Nalgene)
  • 50‐ml polyallomer centrifuge tubes (Nalgene)
  • Econo‐Pac 20‐ml disposable polypropylene columns (Bio‐Rad)
  • 50‐ml screw‐top polypropylene tubes (Becton Dickinson Labware)
  • Rotating platform (Cole‐Parmer)
  • 1.6 × 20–cm C16 column (GE Healthcare)
  • Peristaltic pump
  • Fraction collector
  • 0.8‐cm diameter Poly‐Prep 2‐ml disposable polypropylene column (Bio‐Rad)
  • Additional reagents and equipment for SDS‐PAGE and staining of gels (unit 6.1)
CAUTION: Human placenta should be treated as potentially biohazardous; take suitable precautions, such as wearing latex gloves, eye protection, and a laboratory coat. The homogenization should be performed in a primary cell culture cabinet and any spillage of homogenate or extract should be treated with 1% Virkon. Centrifuge bottles and tubes should be soaked in 1% Virkon after use.

Support Protocol 2: Coupling of Antibodies to Sepharose

  Materials
  • Antibodies/IgG to be coupled: anti‐β1 and anti‐α5 mAbs (e.g., mAb 13 and mAb 16) and rat IgG (Sigma‐Aldrich)
  • Coupling buffer: 0.5 M NaCl/0.1 M NaHCO 3 (prepare fresh)
  • CNBr‐activated Sepharose (Sigma‐Aldrich)
  • 1 mM HCl
  • 1 M ethanolamine in H 2O (store up to 6 months at room temperature)
  • Acetate wash buffer: 0.1 M sodium acetate, pH 4 (adjust with glacial acetic acid)/0.5 M NaCl (store up to 6 months at room temperature)
  • Tris wash buffer: 0.1 M Tris⋅Cl, pH 8 ( appendix 2A)/0.5 M NaCl (store up to 6 months at room temperature)
  • Phosphate‐buffered saline (PBS; prepare using 10× stock solution from Life Technologies) containing 0.05% (w/v) sodium azide (add from 20% sodium azide stock solution in H 2O)
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • Büchner funnel with medium‐porosity fritted‐glass disc
  • Conical flask with side arm
  • 50‐ml screw‐top polypropylene centrifuge tubes
  • Rotating platform (Cole‐Parmer)
  • Additional reagents and equipment for dialysis ( appendix 33)

Support Protocol 3: Biotinylation of Integrin Ligands

  Materials
  • Ligand of interest
  • Coupling buffer 0.5 M NaCl/0.1 M NaHCO 3 (prepare fresh)
  • Sulfo‐NHS Biotin (Pierce)
  • Tris/saline: 25 mM Tris⋅Cl (pH 7.4)/150 mM NaCl
  • Tris/saline containing 0.05% sodium azide
  • Rotating platform (Cole‐Parmer)
  • Additional reagents and equipment for dialysis ( appendix 33) and protein assay ( appendix 33)
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Figures

Videos

Literature Cited

Literature Cited
   Akiyama, S.K., Yamada, S., Chen, W.‐T., and Yamada, K. 1989. Analysis of fibronectin receptor function with monoclonal antibodies: Roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J. Cell Biol. 109:863‐875.
   Byron, A., Humphries, J.D., Askari, J.A., Craig, S.E., Mould, A.P., and Humphries, M.J. 2009. Anti‐integrin monoclonal antibodies. J. Cell Sci. 122:4009‐4011.
   Charo, I.F., Nannizzi, L., Phillips, D.R., Hsu, M.A., and Scarborough, R.M. 1991. Inhibition of fibrinogen binding to GP IIb‐IIIa by a GP IIIa peptide. J. Biol. Chem. 266:1415‐1421.
   Diaz‐Gonzalez, F., Forsyth, J., Steiner, B., and Ginsberg, M.H. 1996. Trans‐dominant inhibition of integrin function. Mol. Biol. Cell 7:1939‐11951.
   Mould, A.P., Wheldon, L.A., Komoriya, A., Wayner, E.A., Yamada, K., and Humphries, M.J. 1990. Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin α4β1. J. Biol. Chem. 265:4020‐4024.
   Mould, A.P., Askari, J.A., Craig, S.E., Garratt, A.N., Clements, J., and Humphries, M.J. 1994. Integrin α4β1‐mediated melanoma cell adhesion and migration on vascular cell adhesion molecule‐1 (VCAM‐1) and the alternatively spliced IIICS region of fibronectin. J. Biol. Chem. 269:27224‐27230.
   Mould, A.P., Akiyama, S.K., and Humphries, M.J. 1995a. Regulation of integrin α5β1‐fibronectin interactions by divalent cations: Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+. J. Biol Chem. 270:26270‐26277.
   Mould, A.P., Garratt, A.N., Askari, J.A., Akiyama, S.K., and Humphries, M.J. 1995b. Identification of a novel monoclonal antibody that recognises a ligand‐induced binding site epitope on the β1 subunit. FEBS Lett. 363:118‐122.
   Mould, A.P., Akiyama, S.K., and Humphries, M.J. 1996. The inhibitory anti‐β1 integrin monoclonal antibody 13 recognises an epitope that is attenuated by ligand occupancy: Evidence for allosteric inhibition of integrin function. J. Biol. Chem. 271:20365‐20374.
   Mould, A.P., Askari, J.A., Aota, S., Yamada, K., Irie, A., Takada, Y., Mardon, H.J., and Humphries, M.J. 1997. Defining the topology of integrin α5β1‐fibronectin interactions using inhibitory anti‐α5 and anti‐β1 monoclonal antibodies: Evidence that the synergy sequence of fibronectin is recognised by the amino‐terminal repeats of the α5 subunit. J. Biol. Chem. 272:17283‐17292.
   Mould, A.P., Burrows, L., and Humphries, M.J. 1998. Identification of amino acid residues that form part of the ligand‐binding pocket of integrin α5β1. J. Biol. Chem. 273:25664‐25672.
   Mould, A.P., Koper, E.J., Byron, A., Zahn, G., and Humphries, M.J. 2009. Mapping the ligand‐binding pocket of integrin α5β1 using a gain‐of‐function approach. Biochem. J. 424:179‐89.
   Newham, P., Craig, S.E., Clark, K., Mould, A.P., and Humphries, M.J. 1998. Analysis of ligand‐induced and ligand‐attenuated epitopes on the leukocyte integrin α4β1: VCAM‐1, MAdCAM‐1 and fibronectin induce distinct conformational changes. J. Immunol. 160:4508‐4517.
   Pytela, R., Pierschbacher, M.D., and Ruoslahti, E. 1985. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 40:191‐198.
   Pytela, R., Pierschbacher, M.D., Argraves, S., Suzuki, S., and Ruoslahti, E. 1987. Arginine‐glycine‐aspartic acid adhesion receptors. Methods Enzymol. 144:475‐489.
   Smith, J.W. and Cheresh, D.A. 1988. The Arg‐Gly‐Asp binding domain of the vitronectin receptor. J. Biol. Chem. 263:18726‐18731.
   Wayner, E.A. and Carter, W.G. 1987. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique α and common β subunits. J. Cell Biol. 105:1873‐1884.
   Yamada, K.M. and Yamada, S.S. 1990. Isolation of fibronectin receptors. In Receptor Purification (G. Litwack, ed.) pp. 435‐449. Humana Press, Clifton, N.J.
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