Lee Sunyoung, Mandic Jelena, Van Vliet Krystyn J
Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2007 Jun 5;104(23):9609-14. doi: 10.1073/pnas.0702668104. Epub 2007 May 29.
The binding kinetics between cell surface receptors and extracellular biomolecules is critical to all intracellular and intercellular activity. Modeling and prediction of receptor-mediated cell functions are facilitated by measurement of the binding properties on whole cells, ideally indicating the subcellular locations or cytoskeletal associations that may affect the function of bound receptors. This dual need is particularly acute vis à vis ligand engineering and clinical applications of antibodies to neutralize pathological processes. Here, we map individual receptors and determine whole-cell binding kinetics by means of functionalized force imaging, enabled by scanning probe microscopy and molecular force spectroscopy of intact cells with biomolecule-conjugated mechanical probes. We quantify the number, distribution, and association/dissociation rate constants of vascular endothelial growth factor receptor-2 with respect to a monoclonal antibody on both living and fixed human microvascular endothelial cells. This general approach to direct receptor imaging simultaneously quantifies both the binding kinetics and the nonuniform distribution of these receptors with respect to the underlying cytoskeleton, providing spatiotemporal visualization of cell surface dynamics that regulate receptor-mediated behavior.
细胞表面受体与细胞外生物分子之间的结合动力学对于所有细胞内和细胞间活动至关重要。通过测量全细胞上的结合特性，有助于对受体介导的细胞功能进行建模和预测，理想情况下可指示可能影响结合受体功能的亚细胞位置或细胞骨架关联。对于配体工程以及用于中和病理过程的抗体的临床应用而言，这种双重需求尤为迫切。在此，我们借助功能化力成像来绘制单个受体并确定全细胞结合动力学，这是通过扫描探针显微镜和使用生物分子共轭机械探针的完整细胞分子力谱实现的。我们在活的和固定的人微血管内皮细胞上，针对一种单克隆抗体定量分析血管内皮生长因子受体-2的数量、分布以及缔合/解离速率常数。这种直接受体成像的通用方法同时定量了这些受体相对于潜在细胞骨架的结合动力学和非均匀分布，提供了调节受体介导行为的细胞表面动力学的时空可视化。
