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

主要研究者：Castro，卡洛斯E.建议编号：1351159细胞与其环境之间施加的力在细胞生理行为中起关键作用，包括细胞伸展、滚动和迁移。 这些细胞牵引力（CTF）通过介导细胞与局部环境的物理通信的膜蛋白来施加。 本论文的目的是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。 将使用两种生理学相关过程作为试验床开发和验证该方法。 拟议的工作对阐明细胞功能和指导细胞分选和生物传感等应用的生物医学设备的设计具有重要的广泛影响。 开发的NMFS设计将被广泛分享，以鼓励这项技术的广泛应用。 PI将开发一个生物分子设计和力学研讨会，通过俄亥俄州州立大学（OSU）的推广工作提供给初中和高中学生，重点是代表性不足的人群。 此外，PI将招募代表性不足的学生参加夏季REU课程和他实验室的高级论文研究。 此外，还将成立一个由多学科的第二和第三年学生组成的年度项目团队，参加年度生物分子设计竞赛。最后，PI在机械工程课程中开发了生物分子力学课程，该课程作为技术选修课提供。这里提出的研究将被利用，包括实验室的组成部分，将促进学生教育的相关原则和技术。 总体而言，PI将实施重大活动，重点是来自代表性不足的人群的学生，将本项目的研究与教育和推广相结合。目前的方法来测量CTF主要依赖于监测基板位移，并需要复杂的数学算法和假设的力量的位置（即在焦点粘连），以确定CTF领域。 虽然这些方法提供了有用的洞察净细胞力和细胞-基质相互作用，CTF的单分子细节，特别是在生理上现实的过程中，仍然知之甚少。 此外，缺乏技术来测量由特定的单膜蛋白传递的力。最近的证据表明，力可能在单个受体的功能中起关键作用，例如B细胞受体，其使用机械能来区分不同亲和力的抗原。 拟议的工作旨在开发和实施一个NMFS直接测量的CTF传输的单膜蛋白和蛋白质复合物的迁移和B细胞抗原检测的生理过程中的两项研究。 具体而言，所提出的工作的目的是：1）设计、构建和校准能够与单个膜蛋白和膜蛋白复合物相互作用的NMFS; 2）使用NMFS测量3 T3成纤维细胞在二维（2D）软基底上迁移的牵引力; 3）使用NMFS测量3 T3成纤维细胞在纤维基质中迁移的牵引力;（4）利用纳米纤维素酶研究机械力和抗原亲和力在B细胞抗原检测中的作用。 这项研究将开发，校准和实施NMFS，能够测量单膜蛋白和蛋白质复合物的CTF。这种单分子直接测量装置将用于在3D纤维环境和抗原检测中迁移的细胞过程中进行以前难以处理的CTF测量。结果有望揭示新的分子洞察力的膜蛋白在关键的生物过程中传输。NMFS将使用纳米技术、支架DNA折纸构建，并将整合用于细胞相互作用（即，RGD-整合素结合）和底物相互作用（即，生物素-链霉亲和素）的功能化、具有校准刚度的弹簧以及用于荧光共振能量转移（FRET）变形读数的荧光染料。 将通过进行两组实验对该器械进行确认：1）测量2D基质上和3D纤维环境中成纤维细胞的CTF，以及2）测量抗原检测期间B细胞的施力。这项工作将结合联合收割机活细胞成像，荧光显微镜，单分子FRET，和DNA折纸，以实现新的见解介导的单分子附件的细胞过程。

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.