Deciphering Mechanosignaling Using Multi-fluorescence Biomembrane Force Probe - Project Summary/Abstract This R35 proposal represents a comprehensive program that dives into the understanding of mechanosignaling, while providing training and mentorship to young scientists. The principal investigator (PI) has over a decade of experience in molecular and cellular mechanobiology in the context of both basic science and translational research. A major gap in the current field of cellular and molecular mechanobiology is that the scientific discoveries are difficult to translate to improve disease diagnosis and treatment, which is partially due to the lack of an effective approach to understand the inner works of mechanosignaling. Taking advantage of the R35 grant mechanism, this program will develop a methodology framework that can decipher principles and mechanisms of mechanosignaling at the sub-molecular level, which is achieved by combining a cutting- edge force spectroscopy platform we developed in house—the multi-fluorescence biomembrane force probe (MF-BFP)—with other in vitro and in silico approaches such as molecular and cellular engineering, super- resolution imaging, biophysical modeling, and molecular dynamics simulation. Integrins, an important family of cell mechanoreceptors, are selected as the molecular model for study due to their profound importance in general physiology and pathology. Seminal concepts under two themes will be explored. Firstly, under the theme of single receptor mechanosignaling, we will study the principles and mechanisms of how single integrins receive, transmit and transduce mechanosignals. We will identify ‘hotspots’ in the integrin structure that mediate integrin mechanosensitivity in ligand binding and conformational changes. By manipulating these hotspots via mutagenesis to directionally and finely tune the integrin mechanosensitivity, we aim to quantitatively manipulate the principles of single integrin mechanosignaling. Secondly, under the theme of receptor cis/trans-crosstalk in mechanosignaling, we will first investigate how two or more integrins cooperate to trigger mechanosignals on a single cell. Next, we will move into a new direction related to the intriguing phenomenon of ‘cell handshaking’, where we will assess the bi-directional biomechanical signal communication between two cells. Collectively, this program will provide in-depth insights into integrin-mediated mechanosignaling and inspire novel strategies for developing integrin-targeting mechanomedicines. Furthermore, the program is anticipated to establish the method and provide the know-how for deciphering mechanosignaling that are applicable to most, if not all, molecular and cellular systems. The widely application of our MF-BFP-centered methodology framework will allow us to better understand mechanobiology-related pathology in various diseases and more efficiently develop mechanobiology-inspired diagnostics and therapeutics in the future. To secure the success of the proposed research, this program will be supported by a strong network of collaborators within and outside of the PI’s home institute.
