title: Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, I: lifetimes of bonds to recombinant alphaLbeta2 under force.
abstract: Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with integrin activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling in leukocytes. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from beta2 integrin in each experiment. Of fundamental importance, the assay for off-rates does not depend on how the force is applied over time, and remains valid when the rates of dissociation change with different levels of force. In this first article, we present results from tests of a monovalent ICAM-1 probe against immobilized alphaLbeta2 in environments of divalent cations (Ca2+, Mg2+, and Mn2+) and demonstrate in detail the method for assay of off-rates. When extrapolated to zero force, the force-free values for the off-rates are found to be consistent with published solution-based assays of soluble ICAM-1 dissociation from immobilized LFA-1, i.e., approximately 10(-2)/s in Mg2+ or Mn2+ and approximately 1/s in Ca2+. At the same time, as expected for adhesive function, we find that the beta2 integrin bonds activated in Mn2+ or Mg2+ possess significant and persistent mechanical strength (e.g., >20 pN for >1 s) even when subjected to slow force ramps (<10 pN/s). As discussed in our companion article, using the same assay, we find that although the rates of dissociation for diICAM-1fc bonds to LFA-1 on neutrophils in Mn2+ are similar to those for mICAM-1 bonds to recombinant alphaLbeta2 on microspheres, they appear to represent a dimeric attachment to a pair of tightly clustered integrin heterodimers. The mechanical strengths and lifetimes of the dimeric interactions increase dramatically when the neutrophils are stimulated by the chemokine IL-8 or are bound with an allosterically activating (anti-CD18) monoclonal antibody, demonstrating the major impact of cell signaling on LFA-1.
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA. evanse@bu.edu
authors.forename: Evan
authors.initials: E
authors.lastname: Evans
authors.forename: Koji
authors.initials: K
authors.lastname: Kinoshita
authors.forename: Scott
authors.initials: S
authors.lastname: Simon
authors.forename: Andrew
authors.initials: A
authors.lastname: Leung
mesh_terms: D001696:Biomechanical Phenomena; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D007700:Kinetics; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D008863:Microspheres; D015335:Molecular Probes / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D013997:Time Factors
chemical_list: D055767:Immobilized Proteins; D016169:Lymphocyte Function-Associated Antigen-1; D015335:Molecular Probes; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1
references.citation: J Leukoc Biol. 2004 Sep;76(3):594-602
references.pmid: 15240749
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15755-60
references.pmid: 18852468
references.citation: Thromb Haemost. 2001 Sep;86(3):746-56
references.pmid: 11583304
references.citation: Methods Cell Biol. 2007;83:373-96
references.pmid: 17613317
references.citation: Biophys J. 2005 Feb;88(2):1458-66
references.pmid: 15556978
references.citation: Cell. 2003 Jan 10;112(1):99-111
references.pmid: 12526797
references.citation: Science. 1978 May 12;200(4342):618-27
references.pmid: 347575
references.citation: Cell. 2002 Sep 20;110(6):673-87
references.pmid: 12297042
references.citation: J Immunol. 2001 Aug 1;167(3):1431-9
references.pmid: 11466362
references.citation: Curr Opin Cell Biol. 2006 Oct;18(5):579-86
references.pmid: 16904883
references.citation: Biomacromolecules. 2006 Nov;7(11):3188-95
references.pmid: 17096550
references.citation: Biophys J. 2010 Apr 21;98(8):1467-75
references.pmid: 20409465
references.citation: Immunity. 2006 Oct;25(4):583-94
references.pmid: 17045822
references.citation: J Immunol. 1998 Jul 15;161(2):836-42
references.pmid: 9670961
references.citation: Biophys J. 2002 Oct;83(4):2270-9
references.pmid: 12324444
references.citation: Annu Rev Cell Dev Biol. 2005;21:381-410
references.pmid: 16212500
references.citation: Biophys J. 1995 Jun;68(6):2580-7
references.pmid: 7647261
references.citation: Nat Immunol. 2005 May;6(5):497-506
references.pmid: 15834409
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):11281-6
references.pmid: 15277675
references.citation: Immunity. 2004 Apr;20(4):393-406
references.pmid: 15084269
references.citation: Biophys J. 2005 Mar;88(3):2288-98
references.pmid: 15653718
references.citation: J Biol Chem. 2005 Aug 5;280(31):28290-8
references.pmid: 15955822
references.citation: Annu Rev Biophys Biomol Struct. 2002;31:485-516
references.pmid: 11988479
references.citation: Immunity. 2007 Jan;26(1):17-27
references.pmid: 17241958
references.citation: Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3784-9
references.pmid: 11274395
references.citation: Annu Rev Immunol. 2007;25:619-47
references.pmid: 17201681