Forces 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.
