Living cells must communicate with each other and with their environment and respond appropriately. This communication is generally mediated by cell surface receptors (CSRs) which receive information in the form of signalling molecules that bind to the receptors (ligands)and respond to that infomation via a signal transduction cascade that leads to a cellular response. Thus, CSRs provide the physical/functional interface between the cell and its environment. It is known that cell phenotype and function are correlative with changes in the cell surface, including the dynamic expression and/or distribution of CSRs. However, the present capacity to track these potential targets and signals of cell function is limited by both biological complexity and a lack of experimental tools. In fact, there exists no current method to quantify this critical signature of cell state at the level of single cells within heterogeneous populations, as a function of real-time response to stimuli, and with molecular resolution. This constraint is illustrated by the increasingly frequent use of combinatorial approaches, high-throughput screening.to find cell types, biochemical factors, and synthetic substrata that result in the desired cell response. From a bioengineering perspective, there is much that could be done to benefit society (e.g., tissue and targeted drug delivery engineering) if we had the capacity to observe dynamic cell-cell and cell-substrate interactions in the context of CSR presence, distribution, and function. From a basic fundamental perspective, there are many important but as-yet-unanswerable questions about cell surface receptor organization and function and how these effect signal transduction mechanisms that could be addressed if we had the capacity to observe these things simultaneously, in real time, with molecular resolution. Dr. VanVliet suggests that such knowledge and the concurrent goal of engineered cell function will be enabled via biofunctionalized nanomechanical imaging of the CSR landscape on living cells. This approach, if successful, will enable unparalleled access to real-time changes in the molecular profile of living cell surfaces, as a function of systematic variation of genetic, biochemical and mechanical stimuli. Dr. VanVliet has previously demonstrated, via collaborative investigations of nanomechanical instabilities in inorganic crystals, that dynamic structural adaptations within materials can be measured, induced, and predicted via contact deformation of surfaces at atomistic/molecular length scales. In this Nanoscale Exploratory Research project, these powerful capabilities will be developed for dynamic living cell surfaces, in order to extend fundamental understanding of the CSR interface through quantitative, simultaneous structural and biochemical imaging via nanomechanical contact.
The objective is to capitalize on the potential of scanning probe microscopy and molecular force spectroscopy to develop a powerful new in vitro cell surface imaging approach through direct, reversible binding between a chemically functionalized mechanical probe and the living cell surface. This approach, termed functionalized force imaging (FFI), will quantify the real-time spatial and temporal distribution of CSRs and internal but near-surface cytoskeletal structure. FFI presents several distinct advantages over existing approaches for CSR analysis: This contact-based imaging does not require cleavage of the cell from its substratum, is not restricted to the analysis of proteins for which purified antibodies are available, and reveals the distribution and relative binding affinity of CSRs that can be tracked over time within individual cells. This project focuses on the development of two specific aspects critical to the feasibility of nanomechanical CSR mapping in living cells: (1) rapid, spatially biased acquisition of both intermolecular forces and structural/mechanical surface profiles; (2) validation of receptor binding specificity in a model cell system for which the number of CSRs per cell can be controlled and quantified through complementary experimental techniques. These objectives will enable the PI.s longer-term goals which include identifying mesenchymal stem and progenitor cell-specific CSR profiles, and then tracking and guiding the differentiation of these cells through external mechanical stimuli for applications ranging from tissue engineered vasculature to chemomechanical sensors.
The intellectual merit of the research will include a new, quantitatively rigorous experimental tool that will catalyze innovative, cross-disciplinary investigations of chemomechanical coupling in biological systems. This broader impact of this project includes a new laboratory-based course on nanomechanical biological experimentation that will be offered to undergraduates of MIT, Wellesley College, and Brandeis University, and that will be co-instructed by the graduate research assistant participating in the above research objectives. This proposal addresses the Biosystems at the Nanoscale research and education theme of the NSF NES solicitation.
