Measurement of 2D receptor-ligand binding kinetics under flow

1159823 Konstantopoulos的智力优势：细胞粘附，通过高度特异性和严格控制的受体-配体（R-L）相互作用介导，在各种生物学事件中起着关键作用。R-L结合的动力学赋予了独特的性质，其允许细胞在生理应力的挑战中彼此相互作用，例如脉管系统中的流体剪切。在生理环境中，受体和它们各自的配体被锚定到并置细胞的表面;因此，R-L结合是一个二维（2D）过程。虽然复杂的生物物理技术最近已经发展到测量无应力的2D亲和力的受体-配体对，他们没有透露其依赖于施加的力1，2。然而，必须阐明这种依赖性，因为与脉管系统中的流体流动相关的力调节介导细胞-细胞粘附的R-L键的动力学。一个综合的实验和数学方法将被开发来确定作为流体动力学剪切的函数的R-L相互作用的二维动力学常数。这种方法利用了R-L结合的物理化学反应的相遇时间的概念。在实验中，将使用光刻和微流体方法来产生以受控的表面密度和取向用蛋白质官能化的长度为L的区域。具有速度U和蛋白质包被区L的流动细胞将与蛋白质相互作用一段相遇时间（<$=L/U），允许从数学建模确定2D受体-配体动力学参数。该模型将充分了解R-L键对施加的剪切力的高度复杂的反应，所有参数都是从伴随的实验中测量的。在概念验证中，实验和分析已经确定了适用于所提出的工作的结果的关键渐近线。本文将研究与胰腺癌转移相关的关键R-L相互作用的二维动力学和微观力学性质。拟议的工作是基础性的，解决了基础科学问题，具有可用于工程设备和治疗干预的潜在结果。本研究的重点是在运输动力学方面的R-L生物物理表征的水平。具体地，将为两个离散的粘合步骤（即，滚动和停滞）分别由不同的R-L对介导：选择素结合粘蛋白16（MUC 16）和足糖萼蛋白样蛋白（PCLP），以及纤维蛋白（原）结合标准形式的CD 44（CD 44 s）。这将通过研究相应的动力学（例如，2D开和关速率）和微机械（例如，使用单分子力谱、微流体和微图案化沿着数学建模来分析上述R-L对的拉伸强度）性质。此外，选择素配体拴系如何促进CD 44 s-纤维蛋白（原）介导的坚定的粘附在剪切应力水平升高的定量理解将被开发。本研究将确定这一系列事件中的限速参数，如选择素或纤维蛋白（原）包被区的长度及其部位密度，这些参数是在升高的剪切应力水平下通过CD 44 s-纤维蛋白（原）结合支持下游牢固粘附所必需的。最后，本项目将确定这些参数是如何通过MUC 16和PCLP的选择性个体敲低来调节的。实验将通过细胞滚动/粘附的计算建模来指导。更广泛的影响：科学/技术：这项工作将推进细胞生物物理学领域的知识。具体来说，这项研究将对渗透生物学的R-L过程的基本科学理解产生广泛的影响。生理学相关环境中R-L介导的细胞粘附动力学的知识将提供设计传感器所需的设计参数，以基于识别靶向生物实体，设计分子以中断不良或病理性粘附事件，例如癌症中的粘附事件，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，在这个项目中开发的实验和数学框架将广泛适用于其他（病理）生理过程中发生的血管。指导女性和代表性不足的学生：欢迎来自外展倡议的学生参与与本研究相关的小型研究项目。(PI的和合作PI的个人联系，通过与巴尔的摩理工高中，宾夕法尼亚大学的ACS项目种子，REU程序）。学生参与：本科生/高中生经常在PI/co-PI的实验室进行研究;通常是女性或来自代表性不足的群体。博士前和博士后指导：博士前和博士后职业发展是PI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.