DYNAMIC STRENGTHS OF SINGLE LEUKOCYTE ADHESION BONDS

单个白细胞粘附键的动态强度

• 批准号: 6328238
• 负责人: EVAN A EVANS
• 金额: $ 35.13万
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6328238
• 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(逐字摘自申请者摘要)：本报告的目的 建议是发展对相互作用的详细生物物理理解 通过定量测定白细胞与内皮细胞的纳米强度 单分子键参与了它们之间的粘合相互作用 细胞。因为相互作用必须在体内发生，在一个大的 流体力学压力，一系列黏附受体@选择素， 整合素和免疫球蛋白(LG)超家族受体参与 胶粘剂相互作用的启动和随后的加强。而当 这些不同的受体在黏附功能中的作用一直很好。 经鉴定，机械强度、时间和化学成分之间的相互作用 在单个分子水平上的单个粘附性相互作用主要是 未知。本提案试图弥合我们在理解上的这一差距 通过使用研究人员最近使用的一种实验策略 被开发用于检测分子连接和测量单键作用力。三 提出了具体的目标来检验关于动力学的各种假设 白细胞与血管内皮细胞的黏附。1)探索以下假设： 每个受体-配体相互作用都是为了满足不同的动态负荷而设计的 在体内发现的要求，研究人员将测量内在的 单键机械强度与断裂时间的关系 涉及多个数量级的选择素和整合素受体 比例。2)检验以下假设：支配债券的显著因素 强度来自小分子成分的独特排列， 研究人员将比较选择素和选择素的动态强度特性 整合素与位点特异性配体的相互作用 通过翻译后修饰和突变进行了改造。3)至 研究粘附力中的分子强度取决于 受体与细胞骨架结构连接的化学以及 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 在细胞膜上原位。这些目标的成功实现是 严重依赖于超灵敏测力探头的使用 超乎寻常的动态范围，可感知分子连接并测量单个 在超脱的巨大时间跨度上的结合力。生物膜 调查人员最近开发的武力探测器BFP满足了这些要求 要求。这种创新的传感器可以以纳米级的精度进行定位 并能量化最弱的非共价键的作用力( 约0.1pN)直到共价键的强度(&gt；1000pN)。同样 本质上，BFP可以在一年内对单分子连接施加压力并使其破裂 脱离时间跨度为六个数量级。通过装饰探头尖端 通过合成和重组配体，研究人员计划测量 驻留在选择素和整合素上的附着的动态强度 中性粒细胞膜，并在玻璃微球上重组。这些研究 将为血管提供令人兴奋的新的和新的生物物理见解 分子水平上的细胞黏附，这将对我们的 了解受体介导的黏附在正常免疫功能和正常免疫功能中的作用 炎症、组织损伤和肿瘤细胞转移的病理生理学。