Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, I: Lifetimes of Bonds to Recombinant αL β2 Under Force
Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, I: Lifetimes of Bonds to Recombinant αL β2 Under Force
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DOI：
10.1016/j.bpj.2009.09.067
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发表时间：
2010-04-21
影响因子：
3.4
通讯作者：
Leung, Andrew
中科院分区：
文献类型：
--
作者：
Evans, Evan;Kinoshita, Koji;Leung, Andrew
使用单分子力光谱探测ICAM-1与固定在微球上的重组α（L）β（2）和中性粒细胞上的β（2）整联蛋白的相互作用，我们量化了令人印象深刻的整联蛋白键的长寿命，高强度状态的层次结构，从二价阳离子溶液中整联蛋白活化的基础水平开始，并随着白细胞中细胞信号传导而急剧上升到超活化状态。利用非常罕见的事件，我们使用在力的稳定斜坡下的键寿命的重复测量，以在每个实验中实现ICAM-1从β（2）整联蛋白的解离速率的直接测定。至关重要的是，解离速率的测定不取决于力如何随时间推移而施加，并且当解离速率随不同水平的力而变化时仍然有效。在这第一篇文章中，我们介绍了在二价阳离子（Ca 2+，Mg 2+和Mn 2+）环境中，单价ICAM-1探针对固定化α（L）β（2）的测试结果，并详细说明了解离速率的测定方法。当外推至零力时，发现解离速率的无力值与公开的可溶性ICAM-1从固定的LFA-1解离的基于溶液的测定一致，即，在Mg 2+或Mn 2+中类似于10（-2）/s，在Ca 2+中类似于1/s。同时，正如对粘附功能的预期，我们发现在Mn 2+或Mg 2+中活化的β（2）整联蛋白键具有显著和持久的机械强度（例如，>20 pN，持续>1 s），即使在缓慢的力斜坡下（
Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alpha(L) beta(2) immobilized on microspheres and beta(2) 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 beta(2) 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 alpha(L) beta(2) 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., similar to 10(-2)/s in Mg2+ or Mn2+ and similar to 1/s in Ca2+. At the same time, as expected for adhesive function, we find that the beta(2) 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 (