Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, I: lifetimes of bonds to recombinant alphaLbeta2 under force.
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.
Evans
Evan
E
Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA. evanse@bu.edu
Kinoshita
Koji
K
Simon
Scott
S
Leung
Andrew
A
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
D055767:Immobilized Proteins; D016169:Lymphocyte Function-Associated Antigen-1; D015335:Molecular Probes; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1
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