CAREER: A Molecular Force Sensor for Single Molecule Studies of Cellular Force Application

PI: Castro, Carlos e .提案号：1351159细胞与其环境之间的作用力在细胞的生理行为中起着关键作用，包括细胞的扩散、滚动和迁移。这些细胞牵引力（CTF）通过膜蛋白施加，介导细胞与局部环境的物理通讯。本课题旨在开发和实现一种纳米级分子力传感器（NMFS），以直接测量单膜蛋白和蛋白复合物的CTF传输。该方法将使用两个生理学相关过程作为测试平台来开发和验证。所提出的工作对阐明细胞功能和指导生物医学设备的设计具有重要的广泛影响，如细胞分选和生物传感。开发的NMFS设计将广泛共享，以鼓励该技术的广泛应用。PI将开发一个生物分子设计和力学讲习班，通过俄亥俄州立大学（OSU）的推广工作向初高中学生提供，重点关注代表性不足的人群。此外，PI将招募代表性不足的学生参加夏季REU项目和在他的实验室进行高级论文研究。还将建立一个由参加年度生物分子设计竞赛的多学科二年级和三年级学生组成的年度项目团队。最后，PI在机械工程课程中开发了生物分子力学课程，作为技术选修课提供。这里提出的研究将包括一个实验室组成部分，这将促进学生在相关原理和技术方面的教育。总体而言，PI将执行重要的活动，重点是来自代表性不足人口的学生，这些活动将该项目的研究与教育和外展结合起来。目前测量CTF的方法在很大程度上依赖于监测基底位移，并且需要复杂的数学算法和关于力的位置（即在焦点粘附处）的假设来确定CTF场。虽然这些方法提供了对净细胞力和细胞-底物相互作用的有用见解，但CTF的单分子细节，特别是在生理现实过程中，仍然知之甚少。此外，还缺乏测量特定单膜蛋白传递力的技术。最近的证据表明，力可能在单个受体的功能中发挥关键作用，例如B细胞受体，它利用机械能区分不同亲和力的抗原。本工作旨在开发和实现一种NMFS，在两项研究中直接测量单膜蛋白和蛋白复合物的CTF传输，重点研究迁移和B细胞抗原检测的生理过程。具体来说，本研究的目标是：1)设计、构建和校准能够与单膜蛋白和膜蛋白复合物相互作用的NMFS；2)利用NMFS测量3T3成纤维细胞在二维（2D）软基质上迁移的牵引力；3)利用NMFS测量3T3成纤维细胞在纤维基质中迁移的牵引力；4)利用NMFS研究机械力和抗原亲和力在B细胞抗原检测中的作用。本研究将开发、校准和实施一种能够测量单膜蛋白和蛋白复合物CTF的NMFS。这种单分子直接测量装置将用于在三维纤维环境和抗原检测的细胞迁移过程中对CTF进行以前难以处理的测量。研究结果有望揭示关键生物过程中膜蛋白力传递的新分子见解。NMFS将使用纳米技术、支架DNA折纸构建，并将整合细胞相互作用（即rgd -整合素结合）和底物相互作用（即生物素-链亲和素）的功能化、校准刚度的弹簧和荧光共振能量转移（FRET）变形读数的荧光染料。该装置将通过两组实验进行验证：1)在2D底物和3D纤维环境中测量成纤维细胞的CTF，以及2)在抗原检测期间测量B细胞施加的力。这项工作将结合活细胞成像、荧光显微镜、单分子FRET和DNA折纸，以获得对单分子附着介导的细胞过程的新见解。

PI: Castro, Carlos E.Proposal Number: 1351159Forces applied between cells and their environment play a critical role in cellular physiologic behavior, including cell spreading, rolling and migration. These cellular traction forces (CTF) are applied via membrane proteins that mediate physical communication of cells with the local environment. The proposed work aims to develop and implement a nanoscale molecular force sensor (NMFS) to directly measure the CTF transmission of single membrane proteins and protein complexes. This methodology will be developed and validated using two physiologically relevant processes as test beds. The proposed work has significant broader impacts on elucidating cellular function and on guiding the design of biomedical devices for applications such as cell sorting and biological sensing. The developed NMFS designs will be broadly shared to encourage widespread application of this technology. The PI will develop a biomolecular design and mechanics workshop to be offered through Ohio State University (OSU) outreach efforts extending to middle and high school students focusing on underrepresented populations. Furthermore, the PI will recruit underrepresented students to participate in both summer REU programs and upper level thesis research in his lab. A yearly project team consisting of multi-disciplinary 2nd and 3rd year students participating in an annual Biomolecular Design Competition will also be established. Finally, the PI has developed a Biomolecular Mechanics course in the Mechanical Engineering curriculum, which is offered as a technical elective. The research proposed here will be leveraged to include a laboratory component that will promote student education in the relevant principles and techniques. Overall, the PI will implement significant activities, with an emphasis on students from underrepresented populations, which integrate the research of this project with education and outreach.Current approaches to measure CTF largely rely on monitoring substrate displacements and require complex mathematical algorithms and assumptions regarding the location of the forces (i.e. at focal adhesions) to determine CTF fields. While these approaches have provided useful insight into net cellular forces and cell-substrate interactions, the single molecule details of CTF, in particular in physiologically realistic processes, remain poorly understood. Furthermore, technology is lacking to measure forces transmitted by specific single membrane proteins. Recent evidence has shown that forces may play a critical role in the function of individual receptors, such as the B cell receptor, which uses mechanical energy to differentiate antigens of varying affinities. The proposed work aims to develop and implement an NMFS to directly measure the CTF transmission of single membrane proteins and protein complexes in two studies focused on the physiological processes of migration and B cell antigen detection. Specifically, the aims of the proposed work are to: 1) design, build and calibrate a NMFS capable of interacting with single membrane proteins and membrane protein complexes; 2) employ the NMFS to measure traction forces of 3T3 fibroblasts migrating on two-dimensional (2D) soft substrates; 3) employ the NMFS to measure traction forces of 3T3 fibroblasts migrating in a matrix of fibers; and 4) employ the NMFS to study the role of mechanical forces and antigen affinity during B cell antigen detection. This research will develop, calibrate, and implement a NMFS that is capable of measuring CTF of single membrane proteins and protein complexes. This single molecule direct measurement device will be implemented to make previously intractable measurements of CTF in the cellular processes of migration in 3D fibrous environments and antigen detection. Results are expected to reveal new molecular insights into force transmission of membrane proteins during critical biological processes. The NMFS will be constructed using the nanotechnology, scaffolded DNA origami, and will integrate functionalization for cellular interaction (i.e. RGD-integrin binding) and substrate interaction (i.e. biotin-streptavidin), springs with calibrated stiffness, and fluorescent dyes for Fluorescence Resonance Energy Transfer (FRET) deformation readouts. The device will be validated by performing two sets of experiments: 1) measuring CTF of fibroblasts on 2D substrates and in 3D fibrous environments, and 2) measuring force application of B cells during antigen detection. This work will combine live cell imaging, fluorescence microscopy, single molecule FRET, and DNA origami to achieve new insights into cellular processes mediated by single molecule attachments.