Friday, May 9, 2025
5/9/2025
In Situ Force Mapping to See Mechanical Communication in Action in Genomes of Living Cells - Project Summary/Abstract Forces are “bits” of mechanical information that cells are thought to use to rapidly (>10 µm/sec) distribute regulatory signals or orchestrate transitions between different functional architectures. Dysregulation of mechanical communication in various subcellular structures is associated with disease including fibrotic disorders, cancers, and cardiovascular pathology, and as such represents a critical target for an emerging class of molecular mechanotherapies. However, our knowledge of whether and how cells process internally and externally generated mechanical cues is still in its infancy. In large part, this is because the current technology has a very limited ability to visualize and quantify intracellular forces originating from ~50-nm sources and spreading micron-scale distances, the information that is critical for understanding the mechanics of large subcellular structures such as the genome and cytoskeleton. To address this challenge, my lab is developing two force nanoprobes that can be delivered to the cell to form a stress-sensitive meshwork capable of reporting forces acting over 18-75-nm range (the size of the probes) and distributed in time and space, thus providing a map of intracellular forces in their normal context. (1) The first nanoprobe provides a direct quantitative measure of force by converting applied mechanical stress to photons, i.e. producing mechanoluminescence. Since no photoexcitation is needed, autofluorescence background and phototoxicity are avoided, providing an unprecedented level of sensitivity, which is unachievable in photoluminescence- based techniques. In this approach, we exploit our recent discovery of the nanoscale mechanism for elastic, repeatable mechanoluminescence in ZnMnS. Force nanoprobes will be based on ZnMnS nanorods with stacking faults, a structural defect, which our research identified as vital for mechanoluminescence. (2) The second nanoprobe is based on dynamic DNA nanostructures, which report applied force by switching between open and closed states with different colors of photoexcited luminescence, i.e. produce a binary readout. The readout is based on the base pairing of a 20-bp DNA sequence that allows for sensitivity in the sub-pN range. Thus, this nanoprobe can register mechanically induced changes in its immediate environment on the scale just above the thermal bath. Finally, we will integrate our force nanoprobes with super-resolution imaging capabilities existing in the lab to create a map of intracellular forces generated by the genome of living HeLa cells. This rich experimental setup will empower us to answer a range of open questions in the field of genome mechanobiology, with the long-term goal to resolve the principles underlying mechanical communication in the 3D genome. Overall, the proposed research program will provide a quantitative method for studying mechanical – and not biochemical – signaling in large subcellular structures in physiological environments. The proposed tools can be translated to clinical samples and thus have the potential to inspire categorically new therapies for diseases associated with pathological mechanotransduction.
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