DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells
| Autor: | Khalid Salaita, Rong Ma, Aaron T. Blanchard, Brendan R Deal, Yuesong Hu, Anna V. Kellner |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Materials science
General Immunology and Microbiology Extramural Tension (physics) General Chemical Engineering General Neuroscience Mice Transgenic DNA Article General Biochemistry Genetics and Molecular Biology DNA metabolism Mechanobiology chemistry.chemical_compound Mice chemistry Nucleic acid Biophysics Animals Transient (computer programming) Dna hairpin |
| Zdroj: | J Vis Exp |
| ISSN: | 1940-087X |
| DOI: | 10.3791/62348-v |
| Popis: | Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions. |
| Databáze: | OpenAIRE |
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