RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes
Abstract 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 t...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-04-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-38157-6 |
| _version_ | 1797836330863951872 |
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| author | Matthew R. Pawlak Adam T. Smiley Maria Paz Ramirez Marcus D. Kelly Ghaidan A. Shamsan Sarah M. Anderson Branden A. Smeester David A. Largaespada David J. Odde Wendy R. Gordon |
| author_facet | Matthew R. Pawlak Adam T. Smiley Maria Paz Ramirez Marcus D. Kelly Ghaidan A. Shamsan Sarah M. Anderson Branden A. Smeester David A. Largaespada David J. Odde Wendy R. Gordon |
| author_sort | Matthew R. Pawlak |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-09T15:08:12Z |
| format | Article |
| id | doaj.art-1fd6216c1397488692642b2a66cb4e0c |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-04-09T15:08:12Z |
| publishDate | 2023-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-1fd6216c1397488692642b2a66cb4e0c2023-04-30T11:21:09ZengNature PortfolioNature Communications2041-17232023-04-0114111110.1038/s41467-023-38157-6RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probesMatthew R. Pawlak0Adam T. Smiley1Maria Paz Ramirez2Marcus D. Kelly3Ghaidan A. Shamsan4Sarah M. Anderson5Branden A. Smeester6David A. Largaespada7David J. Odde8Wendy R. Gordon9Departments of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaDepartments of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaDepartments of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaDepartments of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaDepartment of Biomedical Engineering, University of MinnesotaDepartment of Biomedical Engineering, University of MinnesotaDepartment of Pediatrics, University of MinnesotaDepartment of Pediatrics, University of MinnesotaDepartment of Biomedical Engineering, University of MinnesotaDepartments of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaAbstract 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.https://doi.org/10.1038/s41467-023-38157-6 |
| spellingShingle | Matthew R. Pawlak Adam T. Smiley Maria Paz Ramirez Marcus D. Kelly Ghaidan A. Shamsan Sarah M. Anderson Branden A. Smeester David A. Largaespada David J. Odde Wendy R. Gordon RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes Nature Communications |
| title | RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes |
| title_full | RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes |
| title_fullStr | RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes |
| title_full_unstemmed | RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes |
| title_short | RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes |
| title_sort | rad tgts high throughput measurement of cellular mechanotype via rupture and delivery of dna tension probes |
| url | https://doi.org/10.1038/s41467-023-38157-6 |
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