DYNAMIC STRENGTHS OF SINGLE LEUKOCYTE ADHESION BONDS

单个白细胞粘附键的动态强度

• 批准号: 6711070
• 负责人: EVAN A EVANS
• 金额: $ 32.6万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-10 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6711070
• 关键词: biophysics; cell cell interaction; covalent bond; fibronectins; gene mutation; human tissue; integrins; leukocyte adhesion molecules; ligands; microcapsule; molecular dynamics; neutrophil; posttranslational modifications; selectins; vascular endothelium

DESCRIPTION (Verbatim from Applicant's Abstract): The objective of this proposal is to develop a detailed biophysical understanding of the interactions of leukocytes with endothelial cells by quantitating the nanoscale strengths of single molecular bonds involved in the adhesive interactions between these cells. As the interactions have to occur in vivo in an environment of large hydrodynamic stresses, a repertoire of adhesive receptors @ selectins, integrins and immunoglobulin (lg) super family receptors @ are involved in initiation and subsequent strengthening of the adhesive interaction. While the roles of these different receptors in the adhesive function have been well identified, the interplay between mechanical strength, time, and chemistry of the individual adhesive interactions at the single molecular level is largely unknown. The present proposal attempts to bridge this gap in our understanding by using an experimental strategy that the investigators have recently developed to sense molecular attachments and measure single bond forces. Three specific aims are proposed to test various hypotheses regarding the dynamics of leukocyte adhesion to vascular endothelium. 1) To explore the hypothesis that each receptor-ligand interaction is designed to meet different dynamic loading requirements found in vivo, the investigators will measure the intrinsic relations between mechanical strength and rupture time for single bonds involving selectin and integrin receptors over many orders of magnitude in time scale. 2) To test the hypothesis that the prominent factors governing bond strength arise from unique arrangements of small molecular components, the investigators will compare dynamic strength properties for selectin and integrin interactions with site-specific ligands and ligands that have been engineered through post-translational modification and mutation. 3) To investigate the hypothesis that molecular strength in adhesion depends on chemistry of receptor linkages to cytoskeletal structure as well as ligand-receptor chemistry, the investigators will measure mechanical strengths as a function of rupture times for biofunctional ligand bonds to receptors in situ on cell membranes. The successful accomplishment of these aims is critically dependent on the use of an ultrasensitive force probe with exceptional dynamic range to sense molecular attachments and measure single bond forces over an enormous span of time frame for detachment. The biomembrane force probe BFP, which the investigators have recently developed, meets these requirements. This innovative sensor can be positioned with nanoscale precision and can quantitate forces from the weakest strength of noncovalent bonds ( about0.1 pN) up to the strength of covalent bonds (> 1000pN). Equally essential, the BFP can stress and rupture single molecular attachments over a span of six orders of magnitude in detachment time. By decorating the probe tip with synthetic and recombinant ligands, the investigators plan to measure the dynamic strengths of attachments to selectins and integrins resident on neutrophil membranes and reconstituted on glass microspheres. These studies will provide exciting new and novel biophysical insights into blood-vascular cell adhesion at the molecular level, which will contibute significantly to our understanding of receptor-mediated adhesion in normal immune function and in pathophysiology of inflammation, tissue injury and tumor cell metastasis.

描述（来自申请人摘要的逐字描述）： 建议是发展一个详细的生物物理理解的相互作用 白细胞与内皮细胞的纳米级强度定量 单分子键参与这些之间的粘合剂相互作用 细胞由于相互作用必须在体内大环境中发生， 流体动力学应力，粘附受体@选择素， 整合素和免疫球蛋白（Ig）超家族受体参与了 引发并随后加强粘合剂相互作用。而 这些不同的受体在粘附功能中的作用已经被很好地 确定，机械强度，时间和化学之间的相互作用， 单个分子水平上的单个粘附相互作用在很大程度上 未知目前的建议试图弥合我们理解上的这一差距 通过使用一种实验策略， 用来检测分子附着和测量单键力。三 提出了具体的目标，以测试各种假设的动态 白细胞粘附于血管内皮。1)为了探索这个假设， 每个受体-配体相互作用被设计成满足不同的动态负载 在体内发现的要求，研究人员将测量内在的 单键的机械强度与断裂时间的关系 涉及选择素和整合素受体在时间上的许多数量级 规模2)为了验证这个假设，即支配债券的主要因素 强度来自于小分子成分的独特排列， 研究人员将比较动态强度特性， 整合素与位点特异性配体和已被 通过翻译后修饰和突变来改造。3)到 研究粘附中的分子强度取决于 受体与细胞骨架结构的化学连接以及 配体-受体化学，研究人员将测量机械强度 作为生物功能配体与受体结合的断裂时间的函数， 原位在细胞膜上。这些目标的成功实现， 严重依赖于超灵敏力探针的使用， 卓越的动态范围，可检测分子附着并测量单个 在一个巨大的时间跨度内分离的结合力。生物膜 研究人员最近开发的力探针BFP符合这些要求， 要求.这种创新的传感器可以以纳米级的精度定位 并且可以从非共价键的最弱强度定量力（ 约0.1 pN）至共价键强度（> 1000 pN）。同样 重要的是，BFP可以应力和破坏单分子连接超过一个 分离时间跨度为六个数量级。通过装饰探针尖端 利用合成和重组配体，研究人员计划测量 与选择素和整合素结合的动态强度 中性粒细胞膜并在玻璃微球上重构。这些研究 将提供令人兴奋的新的和新颖的生物物理学见解， 细胞粘附在分子水平上，这将有助于显着我们 了解受体介导的粘附在正常免疫功能和 炎症、组织损伤和肿瘤细胞转移的病理生理学。