Abstract
Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant αL β2immobilized on microspheres and β2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. 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 β2 integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant αL β2 on microspheres in millimolar solutions of divalent cations (Ca2+, Mg2+, Mn2+). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to β2 integrin on the cell surface. Even though ICAM-1 bonds to recombinant αL β2 on microspheres in Mg2+ or Mn2+ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg2+ plus the chemokine IL-8 (I.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg2+ or Mn2+ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bondlike statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1467-1475 |
| Number of pages | 9 |
| Journal | Biophysical Journal |
| Volume | 98 |
| Issue number | 8 |
| DOIs | |
| State | Published - Apr 21 2010 |
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ASJC Scopus subject areas
- Biophysics
Cite this
Long-lived, high-strength states of ICAM-1 bonds to β2 integrin, II : Lifetimes of LFA-1 bonds under force in leukocyte signaling. / Kinoshita, Koji; Leung, Andrew; Simon, Scott; Evans, Evan.
In: Biophysical Journal, Vol. 98, No. 8, 21.04.2010, p. 1467-1475.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Long-lived, high-strength states of ICAM-1 bonds to β2 integrin, II
T2 - Lifetimes of LFA-1 bonds under force in leukocyte signaling
AU - Kinoshita, Koji
AU - Leung, Andrew
AU - Simon, Scott
AU - Evans, Evan
PY - 2010/4/21
Y1 - 2010/4/21
N2 - Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant αL β2immobilized on microspheres and β2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. 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 β2 integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant αL β2 on microspheres in millimolar solutions of divalent cations (Ca2+, Mg2+, Mn2+). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to β2 integrin on the cell surface. Even though ICAM-1 bonds to recombinant αL β2 on microspheres in Mg2+ or Mn2+ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg2+ plus the chemokine IL-8 (I.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg2+ or Mn2+ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bondlike statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.
AB - Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant αL β2immobilized on microspheres and β2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. 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 β2 integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant αL β2 on microspheres in millimolar solutions of divalent cations (Ca2+, Mg2+, Mn2+). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to β2 integrin on the cell surface. Even though ICAM-1 bonds to recombinant αL β2 on microspheres in Mg2+ or Mn2+ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg2+ plus the chemokine IL-8 (I.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg2+ or Mn2+ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bondlike statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.
UR - http://www.scopus.com/inward/record.url?scp=77951630040&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77951630040&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2009.12.4316
DO - 10.1016/j.bpj.2009.12.4316
M3 - Article
C2 - 20409465
AN - SCOPUS:77951630040
VL - 98
SP - 1467
EP - 1475
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 8
ER -