Measurement of 2D receptor-ligand binding kinetics under flow

1159823 Konstantopoulos智力优势：通过高度特异和严格控制的受体-配体(R-L)相互作用介导的细胞黏附，在各种生物事件中发挥关键作用。R-L结合的动力学赋予细胞在生理压力的挑战下相互作用的独特性质，例如血管系统中的流体剪切。在生理环境中，受体及其相应的配体被锚定在相对细胞的表面；因此，R-L结合是一个二维(2D)过程。尽管最近发展了复杂的生物物理技术来测量受体-配体对的非应激2D亲和力，但它们未能揭示其对外力的依赖性1，2。然而，这种依赖性必须被阐明，因为与血管内流体流动相关的力调节R-L键介导细胞-细胞黏附的动力学。将发展一种综合的实验和数学方法来确定R-L相互作用的二维动力学常数作为流体动力剪切的函数。这种方法利用相遇时间的概念来描述R-L结合的物理化学反应。在实验中，将使用光刻和微流控方法来创建具有蛋白质功能化的L长带，该带的表面密度和取向受到控制。一个速度为U的流动细胞和一个被蛋白质覆盖的区域L将与蛋白质相互作用一段时间(≈=L/U)，允许2D受体-配体动力学参数通过数学模型确定。该模型将完全由R-L键对外加剪切力的高度复杂的响应来提供信息，所有参数都是从附带的实验中测量的。在概念论证方面，实验和分析确定了适用于拟议工作结果的关键渐近线。本文将研究与胰腺癌转移相关的R-L相互作用的二维动力学和细观力学性质。拟议的工作是根本性的，涉及基础科学问题，具有潜在的成果，可用于工程设备和治疗干预。本研究的重点在于R-L在生物物理水平上的转运动力学表征。具体地说，将为不同的R-L对介导的两个离散的黏附步骤(即滚动和停止)提供机制解释：选择素分别与粘蛋白16(MUC16)和足突蛋白样蛋白(PCLP)结合，以及纤维蛋白(原)与CD44s的标准型(CD44s)结合。这将通过使用单分子力谱、微流体和微图案化以及数学建模来研究上述R-L对的各自的动力学(例如，2D开启和关闭速率)和微观机械(例如，拉伸强度)性能来实现。此外，还将对选择素-配体连接如何在较高剪应力水平下促进CD44s-纤维蛋白(原)介导的牢固粘连的定量理解有所发展。这项研究将确定在这一系列事件中的限速参数，如选择素或纤维蛋白(原)包被区的长度及其位置密度，以支持在高剪应力水平下CD44s-纤维蛋白(原)结合所需的下游牢固粘连。最后，这个项目将确定这些参数是如何通过选择性地单独敲除MUC16和PCLP来调节的。实验将由细胞滚动/粘合的计算模型来指导。更广泛的影响：科学/技术：这项工作将促进细胞生物物理学领域的知识。具体地说，这项研究将对渗透到生物学中的R-L过程的基本科学理解产生广泛的影响。在生理相关的环境中，R-L介导的细胞黏附动力学的知识将提供所需的设计参数，以设计传感器，基于识别来靶向生物实体，设计分子以阻断不利或病理性黏附事件，例如癌症中的黏附事件，以及一般地询问生物事件。这些基本测量将在胰腺癌的特定背景下进行。虽然这项研究的重点是一个具体的例子，但在这个项目中开发的实验和数学框架将广泛适用于血管系统中发生的其他(病理)生理过程。指导女性和代表性不足的学生：将欢迎来自外联倡议的学生参与与这项研究相关的小型研究项目。(派和联合派的私人联系人，通过与巴尔的摩理工高中的莱森，宾夕法尼亚大学的ACS项目种子，REU计划)。学生参与：本科生/高中生定期在国际和平协会/联合国际的实验室进行研究；通常是女性或来自代表人数较少的群体。博士后和博士后指导：博士后和博士后职业发展是PIs实验室的优先事项。

1159823 KonstantopoulosIntellectual Merit: Cell adhesion, mediated via highly specific and tightly controlled receptor-ligand (R-L) interactions, plays a pivotal role in diverse biological events. The kinetics of R-L binding imparts unique properties that allow cells to interact with one another amidst the challenge of physiological stresses, such as fluid shear in the vasculature. In the physiological setting, receptors and their respective ligands are anchored to the surfaces of apposing cells; thus, R-L binding is a two-dimensional (2D) process. Although sophisticated biophysical techniques have recently been developed to measure the unstressed 2D affinity of receptor-ligand pairs, they fail to disclose its dependence on applied force1,2. This dependence must be elucidated, however, as forces associated with fluid flow in the vasculature modulate the kinetics of R-L bonds mediating cell-cell adhesion. An integrated experimental and mathematical approach will be developed to determine the 2D kinetic constants of R-L interactions as a function of hydrodynamic shear. This approach exploits the concept of encounter time for the physicochemical reaction of R-L binding. In experiment, lithographic and microfluidic methods will be used to create zones of length L functionalized with proteins at controlled surface density and orientation. A flowing cell with velocity U and a protein-coated zone L will interact with the protein for an encounter time (ô=L/U), allowing the 2D receptor-ligand kinetic parameters to be determined from mathematical modeling. The model will be fully informed by the highly complex response of R-L bonds to applied shear force, with all parameters measured from accompanying experiments. In proof of concept experiments and analysis have identified key asymptotes that apply to the results of the proposed work. The 2D kinetic and micromechanical properties of critical R-L interactions pertinent to pancreatic cancer metastasis will be studied herein. The proposed work is fundamental, addressing basic science issues, with potential results that can be leveraged in engineering devices and therapeutic interventions. The focus of this study is at the level of R-L biophysical characterization in terms of transport kinetics. Specifically, a mechanistic interpretation will be provided for two discrete adhesion steps (i.e., rolling and arrest) mediated by distinct R-L pairs: selectin binding to mucin 16 (MUC16) and podocalyxin-like protein (PCLP), and fibrin (ogen) binding to the standard form of CD44 (CD44s), respectively. This will be achieved by investigating the respective kinetic (e.g., 2D on- and off- rates) and micromechanical (e.g., tensile strength) properties of the aforementioned R-L pairs using single-molecule force-spectroscopy, microfluidics and micropatterning along with mathematical modeling. Moreover, a quantitative understanding of how selectin-ligand tethering facilitates CD44s-fibrin(ogen) mediated firm adhesion at elevated levels of shear stress will be developed. This study will determine the rate-limiting parameters in this cascade of events, such as the lengths of selectin- or fibrin(ogen)-coated zones and their site densities, necessary to support downstream firm adhesion by CD44s-fibrin(ogen) binding at elevated shear stress levels. Finally, this project will determine how these parameters are modulated by selective individual knockdown of MUC16 and PCLP. Experiments will be guided by computational modeling of cell rolling/adhesion. Broader Impacts: Scientific/ Technological: This work will advance the knowledge in the field of cell biophysics. Specifically, this research will have broad impact in the basic scientific understanding of R-L processes that permeate biology. Knowledge of the kinetics of R-L-mediated cell adhesion in physiologically relevant settings will provide design parameters needed to engineer sensors, to target biological entities based on recognition, to design molecules to interrupt adverse or pathological adhesion events, such as those in cancer, and, generically, to interrogate biological events. These fundamental measurements will be performed in the specific context of pancreatic cancer. While this study focuses on a specific example, the experimental and mathematical framework developed in this project will be broadly applicable to other (patho)physiological processes occurring in the vasculature. Mentoring of Female and Under-represented Students: Students from outreach initiatives will be welcome to work on small research projects associated with this research. (PI's and co-PI's personal contacts, via laison with Baltimore Polytechnic high-school, Penn's ACS Project SEED, REU programs). Student Participation: Undergraduate/high school students regularly perform research in the PI's/co-PI's labs; often women or from underrepresented groups. Pre- and Post-doctoral mentoring: Pre- and post-doctoral career development is a priority in the PIs' laboratories.