From governing the adhesion and recruitment of leukocytes in the immune response to determining cell fate and tissue development, mechanical forces play a key regulatory role throughout biology. This emergent field of """"""""mechanobiology"""""""" is leading to new understandings of disease such as bleeding disorders, cancer, osteoporosis and asthma, as we recognize that mechanics can play a large role in physiological responses at the molecular, cellular and organismic levels. Technological developments that enable precise manipulation of single molecules and cells (e.g. optical tweezers and AFM) have been a driving force in the development of the field. However, growth of the field is impeded by limited access to such technology as it can be expensive, technically challenging, and low-throughput. To overcome these challenges, we will develop a bold approach for applying controlled forces to microscopic samples with single-molecule precision that is inexpensive, simple to use, and high-throughput. By integrating a centrifuge and a microscope, we have demonstrated a prototype Centrifuge Force Microscope (CFM) to perform thousands of single-molecule force experiments in parallel. We propose to take this concept of single-molecule centrifugation to the next level, by developing a powerful, multi- purpose, benchtop instrument for mechanobiology that can be used by a variety of biomedical researchers. We will accomplish this by 1) Enabling massively parallel single-molecule measurements of structural transitions such as protein unfolding by integrating high-resolution imaging into the CFM;2) Increasing the accessibility of single-molecule manipulation techniques by miniaturizing the high-resolution CFM to be incorporated directly into a standard benchtop centrifuge;3) Demonstrating the versatility of this CFM by measuring intra- and inter-molecular bond strengths at both the single-molecule and single-cell levels. In summary, this project will result in an accessible, high-throughput and high-resolution new platform for measuring biological systems under mechanical force at the nanoscale with a broad range of applications, ranging from measuring structural transitions within individual molecules, to measuring the affinity of single cells, to measuring the compliance of soft samples. This instrument will dramatically reduce cost and improve performance and safety by leveraging one of the most common pieces of laboratory equipment - the benchtop centrifuge. This approach will remarkably lower the barrier for researchers to do single- molecule manipulation experiments by requiring little technical expertise and by offering a 1000 fold efficiency boost and a 10-100 fold cost improvement from many other methods. This project is significant since it will open up the fields of mechanobiology and single-molecule manipulation to many new researchers and systems, accelerating the pace of discovery.

Public Health Relevance

Mechanical forces play key regulatory roles in biology, with the emergent field of mechanobiology leading to new understandings of diseases such as bleeding disorders, cancer, osteoporosis and asthma. However, discoveries have been impeded by limited access to instrumentation, due to their expense, challenging technically requirements, and inefficiency. We will solve these problem by developing a new platform that integrates a compact high-resolution microscope into a benchtop centrifuge to offer a 1000 fold improvement in efficiency and a 10-100 fold reduction in cost, opening up the fields of mechanobiology and single-molecule manipulation to many new researchers and systems, and accelerating the pace of discovery.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZGM1-BBCB-A (ID))
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Friedman, Fred K
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Children's Hospital Boston
United States
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Yang, Darren; Wong, Wesley P (2018) Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy. Methods Mol Biol 1665:353-366
Yang, Darren; Wong, Wesley P (2016) Small but Mighty: Nanoparticles Probe Cellular Signaling Pathways. Dev Cell 37:397-8
Yang, Darren; Ward, Andrew; Halvorsen, Ken et al. (2016) Multiplexed single-molecule force spectroscopy using a centrifuge. Nat Commun 7:11026