In Situ Detection of Integrin Ligands

Daiji Kiyozumi1, Ryoko Sato‐Nishiuchi1, Kiyotoshi Sekiguchi1

1 Laboratory of Extracellular Matrix Biochemistry, Institute for Protein Research, Osaka University, Osaka
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 9.7
DOI:  10.1002/0471143030.cb0907s65
Online Posting Date:  December, 2014
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Abstract

Integrins are cell surface receptors for cell adhesion. Integrin‐mediated cell adhesion regulates various cellular processes, including cell survival, migration, proliferation, and differentiation. In vivo, ligands for integrins are immobilized within extracellular matrices, insoluble sheet‐like or fibrous supramolecular complexes that associate with or surround cells. To better understand the molecular basis of integrin‐mediated regulation of cellular behavior in vivo, it is of critical importance to collect information regarding the activities as well as spatial distributions of integrin ligands in situ. This unit describes a protocol for detecting the spatial distribution of the complement of integrin ligands in situ by overlaying soluble recombinant integrins. © 2014 by John Wiley & Sons, Inc.

Keywords: integrin; extracellular matrix (ECM); basement membrane; cell adhesion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Expression and Purification of Recombinant Soluble Integrin α‐β Dimers
  • Support Protocol 1: Construction of Integrin Expression Plasmids
  • Basic Protocol 2: In Situ Binding of Recombinant Integrins
  • Support Protocol 2: Preparation of Dimerized Acid and Base Peptides
  • Support Protocol 3: Preparation of Frozen Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Expression and Purification of Recombinant Soluble Integrin α‐β Dimers

  Materials
  • Freestyle™ 293‐F cells (Life Technologies, cat. no. R790‐07)
  • Freestyle™ 293 Expression Medium (Life Technologies, cat. no. 12338‐018)
  • Sterilized 1‐liter Erlenmeyer flask (e.g., polycarbonate flask, Corning, cat. no. 431147)
  • Integrin expression plasmids (see protocol 2)
  • Opti‐MEM® I Reduced Serum Medium (Life Technologies, cat. no. 11058‐021)
  • Sterilized 15‐ml and 50‐ml polypropylene tubes
  • 293fectin™ Transfection Reagent (Life Technologies, cat. no. 12347‐019)
  • 1 M Tris·Cl, pH 8.0 ( appendix 2A)
  • Tris‐buffered saline (TBS, see recipe)
  • 3 M imidazole stock solution (see recipe)
  • 200 mM phenylmethylsulfonyl fluoride (PMSF)/DMSO (see recipe)
  • Ni‐NTA agarose (Qiagen, cat. no. 30210)
  • Econo‐Column® chromatography column, 1.5 × 10 cm (Bio‐Rad, cat. no. 737‐1512)
  • Polyprep® chromatography column, 0.8 × 4 cm (Bio‐Rad, cat. no. 731‐1550)
  • His wash buffer (see recipe)
  • 6% SDS‐polyacrylamide gel (unit 6.1)
  • Anti‐FLAG M2 affinity gel (Sigma, cat. no. A2220)
  • 5 mg/ml FLAG peptide/TBS (see recipe)
  • Dialysis membrane with a molecular weight cut‐off of 20,000 Da (e.g., Slide‐A‐Lyzer 20K MWCO Dialysis Cassettes, Pierce, cat. no. 66003)
  • Bradford protein assay reagent [e.g., Coomassie (Bradford) Protein Assay Reagent, Pierce, cat. no. 23200]
  • 500‐ml centrifuge bottle
  • Large volume centrifuge rotor (e.g., JA‐10, Beckman Coulter)
  • Large volume centrifuge (e.g., Avanti J‐26S, Beckman Coulter)
  • 1.5‐ml polypropylene tube
  • Rocking platform
  • 500‐ml Erlenmeyer flask
  • Additional reagents and equipment for one‐dimensional SDS gel electrophoresis of proteins (unit 6.1), staining proteins in gels (unit 6.6), and dialysis and concentration of protein solutions ( appendix 3C)

Support Protocol 1: Construction of Integrin Expression Plasmids

  Materials
  • Mammalian expression vector [e.g., pcDNA3.1(+), Life Technologies, cat. no. V790‐20]
  • cDNAs encoding integrin α and β subunits (e.g., cloned integrin cDNAs or reverse transcripts of tissue RNAs)
  • NOTE: See appendix 3A for cross‐references to molecular biology procedures.

Basic Protocol 2: In Situ Binding of Recombinant Integrins

  Materials
  • Frozen tissue sections
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% paraformaldehyde/PBS (4% PFA/PBS; see recipe)
  • Tris‐buffered saline (TBS; see recipe)
  • TBS containing 1 mM MnCl 2 (TBS‐Mn; see recipe)
  • Blocking solution (3% BSA/TBS‐Mn; see recipe)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • Moist chamber
  • Purified rabbit anti‐Velcro polyclonal antibody (see protocol 4)
  • Fluorophore‐conjugated anti‐rabbit IgG [e.g., Alexa Fluor® 488 Goat Anti‐Rabbit IgG (H+L) Antibody, Life Technologies, cat. no. A‐11034]
  • Hoechst 33342 solution (e.g., Hoechst 33342, Thermo Scientific, cat. no. 62249)
  • Mounting medium (e.g., Fluorescence Mounting Medium, Dako, cat. no. S3023)
  • Hair dryer
  • Coverslips
  • Fluorescence microscope
  • Additional reagents and equipment for immunofluorescence staining (unit 4.3)

Support Protocol 2: Preparation of Dimerized Acid and Base Peptides

  Materials
  • HPLC‐purified ACID peptide, NH 2‐GCAQLEKELQALEKENAQLEWELQALEKELAQ‐OH (e.g., commercially synthesized by Bio‐Synthesis Inc.)
  • HPLC‐purified BASE peptide, NH 2‐AQLKKKLQALKKKNAQLKWKLQALKKKLAQ‐OH (e.g., commercially synthesized by Bio‐Synthesis Inc.)

Support Protocol 3: Preparation of Frozen Tissue Sections

  Materials
  • Isopentane
  • Liquid nitrogen
  • OCT compound (e.g., Tissue‐Tek® O.C.TTM Compound, Sakura Finetek, cat. no. 4583)
  • Freshly isolated tissue of interest
  • Pyrex beaker
  • Polystyrene box
  • Tissue mold
  • Plastic zipper bag
  • Cryostat
  • Adhesive‐coated glass slides (e.g., MAS‐GP typeA White, Matsunami, cat. no. S9901)
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Figures

Videos

Literature Cited

Literature Cited
  Dana, N., Fathallah, D.M., and Arnaout, M.A. 1991. Expression of a soluble and functional form of the human beta2 integrin CD11b/CD18. Proc. Natl. Acad. Sci. U.S.A. 88:3106‐3110.
  Denda, S., Reichardt, L.F., and Müller, U. 1998. Identification of osteopontin as a novel ligand for the integrin alpha8beta1 and potential roles for this integrin‐ligand interaction in kidney morphogenesis. Mol. Biol. Cell 9:1425‐1435.
  Hynes, R.O. 2002. Integrins: Bidirectional, allosteric signaling machines. Cell 110:673‐687.
  Ido, H., Harada, K., Yagi, Y., and Sekiguchi, K. 2006. Probing the integrin‐binding site within the globular domain of laminin‐511 with the function‐blocking monoclonal antibody 4C7. Matrix Biol. 25:112‐117.
  Jeong, S.J., Luo, R., Singer, K., Giera, S., Kreidberg, J., Kiyozumi, D., Shimono, C., Sekiguchi, K., and Piao, X. 2013. GPR56 functions together with alpha3beta1 integrin in regulating cerebral cortical development. PLoS One 8:e68781.
  Kiyozumi, D., Takeichi, M., Nakano, I., Sato, Y., Fukuda, T., and Sekiguchi, K. 2012. Basement membrane assembly of the integrin alpha8beta1 ligand nephronectin requires Fraser syndrome‐associated proteins. J. Cell Biol. 197:677‐689.
  Mould, A.P., Akiyama, S.K., and Humphries, M.J. 1995. Regulation of integrin alpha5beta1‐fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+. J. Biol. Chem. 270:26270‐26277.
  Müller, U., Wang, D., Denda, S., Meneses, J.J., Pedersen, R.A., and Reichardt, L.F. 1997. Integrin alpha8beta1 is critically important for epithelial‐mesenchymal interactions during kidney morphogenesis. Cell 88:603‐613.
  Nishiuchi, R., Takagi, J., Hayashi, M., Ido, H., Yagi, Y., Sanzen, N., Tsuji, T., Yamada, M., and Sekiguchi, K. 2006. Ligand‐binding specificities of laminin‐binding integrins: A comprehensive survey of laminin‐integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins. Matrix Biol. 25:189‐197.
  O'Shea, E.K., Lumb, K.J., and Kim, P.S. 1993. Peptide “Velcro”: Design of a heterodimeric coiled coil. Curr. Biol. 3:658‐667.
  Sato, Y., Uemura, T., Morimitsu, K., Sato‐Nishiuchi, R., Manabe, R., Takagi, J., Yamada, M., and Sekiguchi, K. 2009. Molecular basis of the recognition of nephronectin by integrin alpha8beta1. J. Biol. Chem. 284:14524‐14536.
  Sato‐Nishiuchi, R., Nakano, I., Ozawa, A., Sato, Y., Takeichi, M., Kiyozumi, D., Yamazaki, K., Yasunaga, T., Futaki, S., and Sekiguchi, K. 2012. Polydom/SVEP1 is a ligand for integrin alpha9beta1. J. Biol. Chem. 287:25615‐25630.
  Takagi, J., Erickson, H.P., and Springer, T.A. 2001. C‐terminal opening mimics “inside‐out” activation of integrin alpha5beta1. Nat. Struct. Biol. 8:412‐416.
  Takagi, J., DeBottis, D.P., Erickson, H.P., and Springer, T.A. 2002. The role of the specificity‐determining loop of the integrin beta subunit I‐like domain in autonomous expression, association with the alpha subunit, and ligand binding. Biochemistry 41:4339‐4347.
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