RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes.

Autor: Pawlak MR; Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA., Smiley AT; Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA., Ramirez MP; Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA., Kelly MD; Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA., Shamsan GA; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Anderson SM; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Smeester BA; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA., Largaespada DA; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA., Odde DJ; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA., Gordon WR; Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA. wrgordon@umn.edu.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2023 Apr 28; Vol. 14 (1), pp. 2468. Date of Electronic Publication: 2023 Apr 28.
DOI: 10.1038/s41467-023-38157-6
Abstrakt: Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis.
(© 2023. The Author(s).)
Databáze: MEDLINE
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