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 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.

Fig. 1. Overview and validation of RAD-TGT function in CHO-K1 cells.

a Conceptual schematic of the force-induced rupture and readout of RAD-TGTs. b Brightfield imaging at 20x of adhesion assay of cells on RAD-TGT surfaces with either WDV, WDV-FN, or WDV-Echi ligand and 12 or 54 pN rupture force. c Fluorescent imaging at 40x of qTGT fluorescent duplex rupture of cells plated on TGT surfaces of varying ligand and rupture force composition. White dotted lines denote cell borders. d Flow cytometry results of cells plated on RAD-TGT surfaces with different ligand and rupture force composition. e Representative histogram showing the effect of para-amino-blebbistatin treatment on TGT rupture. f SuperPlots of CY5 fluorescence of each cell from three biological replicates normalized to WDV median fluorescence with symbols for medians of biological replicates. Representative histograms turned 90° and scaled linearly for reference. Gray histograms are WDV only, dotted lines are with para-amino-Blebbistatin, and solid orange is with DMSO treatment as control. **p = 0.001387, *p = 0.015441. Statistics were performed using one-way ANOVA of the medians of biological replicates. Source data are provided as a Source Data file. All cartoons and schematics were created with Biorender.com.

Fig. 2. RAD-TGT measurements in U251 cells readout by flow cytometry and sequencing of internalized DNA barcodes.

a SuperPlots of the fold change of CY5 fluorescence intensity of individual U251 cells plated on 12 pN (top) or 54 pN (bottom) RAD-TGTs with either WDV, WDV-FN, or WDV-Echi. Triangles represent the median fold change of population per replicate; the horizontal red line is at the median fold change for all cells analyzed. n = 8 or 5 independent experiments for 12 pN and 54 pN graphs, respectively. ***p = 0.0002, ****p < 0.0001. b Brightfield and qTGT imaging of U251 cells plated on either 12 pN or 54 pN qTGTs with WDV-Echi ligand. c Schematic of 12 pN RAD-TGT with barcode used for sequencing experiments and representative Sanger sequencing trace with each ligand barcode. d Sanger and next-generation sequencing results of biological triplicates of U251 cells plated on an equimolar mixture of 12 pN RAD-TGTs with all three ligands present. **p = 0.0077; ****p < 0.0001. Source data are provided as a Source Data file. All statistics were performed using a one-way ANOVA of the medians of biological replicates. Error bars are standard deviations.

Fig. 3. RAD-TGTs detect CRISPR-KO of mechanosensing proteins in single and mixed cell populations.

a SuperPlots of the fold change of CY5 fluorescence intensity of individual WT, TLN1 KO, or CD44 KO U251 cells on 12 pN (top) or 54 pN (bottom) RAD-TGTs with WDV-Echi. Symbols represent the median Cy5 fold change for each replicate experiment. Identical symbols indicate the samples were collected on the same day. The horizontal red line is at the median fold change for all cells analyzed. For 12 pN experiments, n = 8 (WT), 3 (CD44KO), or 6 (talinKO) and for 54 pN experiments n = 5(WT) and 3 (CD44KO and talinKO). A two-tailed paired t-test was used for statistics, *p = 0.0360 (12 pN CD44KO), **p = 0.0028 (12 pN talinKO), and *p = 0.0322 (54 pN talinKO). Medians were paired based on the day each experiment was performed. b Histogram of a mixed population of U251 WT, Talin1, and CD44 KO (gray solid), overlaid with histograms of cell lines tested individually (outlines only). c Violin plots of individual populations, mixed population, and the sum of individual populations. d Cartoon of U251 WT and CD44 KO cells and accompanying histogram of the two cell types mixed together (gray) or individual. The composition of the population is determined by measuring the GFP signal on different applied gates of the CY5 histogram as CD44 KO also expresses GFP. The composition of the lowest 20%, highest 20%, and the entire population is displayed. e Scatter plots of U251 and CHO-K1 cells plated on surfaces containing a 12 pN Cy5 labeled RAD-TGT and 54 pN A488 labeled RAD-TGT for both each cell individually and in a mixed population. Source data are provided as a Source Data file. All cartoons and schematics were created with Biorender.com.