Mechanical forces play key roles throughout biology, from governing the adhesion of leukocytes in the immune response, to determining cell fate and tissue development. This emergent field of mechanobiology is providing vital insights into diseases such as bleeding disorders, cancer, and infectious diseases, where it is becoming clear that conventional biochemical and genomic characterizations are not sufficient to understand the rich behavior of living systems or how they fail. Rather, we must uncover how force can change the structure and function of molecules, and trigger mechanotransduction pathways to modify cell responses. 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 technologies as they can be expensive, technically challenging, and low- throughput. These challenges have also limited the types of scientific questions that can be addressed. To overcome these challenges, we will develop high-throughput and accessible new approaches in mechanobiology that will (i) open up new areas of study through the introduction of new capabilities, and (ii) democratize single-molecule force measurements so that all biomedical researchers can make discoveries using these powerful tools. For example, we will accelerate single-molecule measurements by building upon an instrument that almost all biomedical researchers already have: the benchtop centrifuge. By developing a miniature microscope that fits into a standard centrifuge bucket, we will create an accessible and inexpensive benchtop instrument that will bring high-throughput single-molecule manipulation to non-specialists, offering a 1000 fold efficiency boost and 10-100 fold cost improvement over many other methods. We will also develop self-assembled DNA nanoscale devices that facilitate single-molecule studies of population heterogeneity, and that enable instrument-free force spectroscopy. Significantly, these projects will open the fields of mechano- biology and single-molecule manipulation to new researchers and systems, accelerating the pace of discovery. Additionally, we will apply our single-molecule approaches to answer key open questions in mechanobiology regarding (i) the mechanical regulation of hemostasis, (ii) adhesion molecules in the immune response, and (iii) mechanotransduction and the molecular basis for hearing and deafness. For example, we will perform massively-parallel force measurements using single-molecule centrifugation to study force-regulated enzymatic cleavage of von Willebrand factor, and investigate mutations related to von Willebrand Disease, the most common inheritable bleeding disorder. We will also study cellular adhesion of leukocytes, and investigate the molecular basis of hearing and deafness. Overall, these efforts should firmly establish force as a key parameter for understanding the basic processes of life, and provide a new handle for both understanding? and treating?disease.

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, and infectious diseases. However, discoveries have been impeded by limited access to instrumentation, due to their expense, challenging technically requirements, and inefficiency, as well as limitations in current capabilities. We will solve these problem by developing powerful and accessible new nanoscale approaches that will democratize the fields of mechanobiology and single-molecule manipulation to accelerating the pace of discovery, and we will apply these approaches to answer key open questions in the areas of hemostasis, immune function, and hearing and deafness.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZRG1)
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Flicker, Paula F
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Boston Children's Hospital
United States
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Nathwani, Bhavik; Shih, William M; Wong, Wesley P (2018) Force Spectroscopy and Beyond: Innovations and Opportunities. Biophys J 115:2279-2285
Yang, Darren; Wong, Wesley P (2018) Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy. Methods Mol Biol 1665:353-366
Hansen, Clinton H; Yang, Darren; Koussa, Mounir A et al. (2017) Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection. Proc Natl Acad Sci U S A 114:10367-10372
Fu, Hongxia; Jiang, Yan; Yang, Darren et al. (2017) Flow-induced elongation of von Willebrand factor precedes tension-dependent activation. Nat Commun 8:324
Yang, Darren; Ward, Andrew; Halvorsen, Ken et al. (2016) Multiplexed single-molecule force spectroscopy using a centrifuge. Nat Commun 7:11026