Project summary The proposed Center on Probes for Molecular Mechanotechnology (CPMM) will work to develop and optimize technologies to enable the study of mechanobiology and mechanotransduction pathways in living cells. The CPMM includes three highly synergistic Technology Development Projects (TDPs) that will be led by Alexa Mattheyses, Khalid Salaita, and Yonggang Ke who have a strong track record of jointly publishing and working together to developed tension probe technologies. In TDP#1: High resolution probes for mechanobiology, we will create “indestructible” probes that can push the limits of spatial and orientation resolution for the DNA tension probe technology. Tension-PAINT imaging will be refined to achieve realtime 20 nm spatial mapping of forces and to combine this with immunostaining to map the proteins that assemble within proximity to mechanically active receptors. Force orientation will be mapped using fluorescence polarization methods with turn-key commercial microscopes. In TDP#2: Probes for mechanical tagging, we will develop methods of force-induced tagging. The central design feature is a DNA probe that mediates a binding event or dissociation event at threshold levels of force. Cells are tagged based on the magnitude and frequency of mechanical events generated by a cell surface receptors. This TDP will lead to high-throughput analysis of cells using flow cytometry and will also allow for proteomic analysis to open the door to “mechanomics”. Under TDP#3: Amplified force sensors, the central technology here is responsive DNA structures that amplify mechanical inputs. The CPMM has nine associated inaugural Driving Biomedical Projects (DBPs) led by a team of geographically diverse collaborators. DBPs #1-#4 are focused on mechanobiology of T cells and use CPMM tools to test the mechanosensor function of the T cell receptor (TCR) and the adhesion receptor LFA-1. DBP#5 focuses on the heterogeneity in cancer cells. DBP#6 and #7 target the mechanosensor responses of platelets. Finally, DBP#8 and #9 address fundamental questions of the role of mechanics in focal adhesions. Our prototype TDP technologies provide methods to measure molecular forces with the same ease and simplicity as that of immunostaining, flow cytometry, PCR and ELISA. But unlike these mainstream techniques, mechano-imaging, mechano-PCR, mechano-flow, and mechano-ELISA are not commercialized. Hence, the reagents and surface preparation protocols and data analysis routines have to be custom prepared by the end user. This can be challenging to the non-expert and is not routine. Therefore, the CPMM will integrate a strong Community Engagement (CE) component. CE activities will focus on hands-on training workshops, publication of methods articles, virtual seminar series, industry engagement, a strong web presence and engagement with three key mechanobiology conferences that will help accelerate adoption of the tension probe technology. These CE activities will ultimately lead to commercialization which will enable wide spread dissemination across the various cell biology communities.

Public Health Relevance The Center proposal aims to develop and disseminate new technologies to measure the mechanical forces in living cells. If successful, these new technologies will reveal the forces that guide cellular processes such as normal and abnormal cell growth, immune responses, and clotting. As such, center technologies will improve our ability to diagnose and treat diseases ranging from cancer metastasis to stroke.