Flow cytometry is used routinely to measure single-cell gene expression by staining cells with fluorescent antibodies and nucleic acids. Here, we present tension-activated cell tagging (TaCT) to label cells fluorescently based on the magnitude of molecular force transmitted through cell adhesion receptors. As a proof-of-concept, we analyzed fibroblasts and mouse platelets after TaCT using conventional flow cytometry.
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A data summary file containing all individual replicate data from the main text, all individual replicate data from the supporting information and all P values from statistical analyses performed in this manuscript is provided at https://osf.io/hkrve/?view_only=cc90b513cb5547358c652e5ace836106. Source data are provided with this paper.
Code used for oxDNA data analysis and graph generation is publicly available online at https://github.com/SalaitaLab/Tension_activated_cell_tagging.
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K.S. acknowledges support from the National Institutes of Health through the National Institute of General Medical Sciences (NIGMS) grants RM1GM145394 and R01GM131099 and the National Institute of Allergy and Infectious Diseases grant no. AI172452.
The authors declare no competing interests.
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(a) Scheme showing that when integrin is in the inactive state, the peeling probe remains in the duplex form. As integrin binds and pulls on the RGD ligand, if F < Fpeel, the peeling strand is intact, and if F > Fpeel, the duplex dehybridizes and the Cy3B fluorescence turns on. FA proteins such as vinculin, talin, and FAK, as well as actin cytoskeleton participate extensively during the integrin force generation and mechanotransduction. (b) Histogram of 227 NIH3T3 cells showing the distribution of average %peel/µm2 per cell after 1 h incubation on peeling probe substrate. Data acquired from 3 biological replicates, bin width = 0.1%. (c) Representative microscopy images show that GFP-vinculin colocalized with integrin tension signal. Images were acquired with MEF GFP-vinculin cells that were cultured on peeling probe substrate (n = 3). (d) Representative microscopy images show that the phosphorylated FAK (pY397) colocalized with fixed integrin tension signal. MEF cells were incubated on peeling probe substrate for 40–45min, fixed and stained with Rabbit anti-FAK pY397 and Alexa488 labeled secondary antibody, followed by imaging (n = 3). (e) Representative microscopy images show that the actin stress fibers colocalized with fixed tension signal (n = 3). MEF cells were incubated on peeling probe substrate for 60–90 min, fixed and stained with Alexa647-phalloidin, followed by imaging.
(a) Schematics comparing the mechanism of TaCT/peeling probe and TGTs. Peeling probe maps integrin tension greater than 41 pN as the BHQ2 strand is separated, and the RGD anchor remains despite the duplex dehybridization. In contrast, TGTs map integrin forces greater than 56 or 12 pN in the shearing or unzipping geometry. The force-induced rupture of the duplex generates a Cy3B turn-on fluorescence signal as the top BHQ2 strand separates from the Cy3B anchor strand. Unlike peeling strand, the loss of the RGD anchors in TGTs terminates mechanotransduction through integrins. (b) Representative microcopy images of NIH3T3 cells incubated on peeling probe or TGT substrates for ~45–60 min (n = 3). Second row of images show the zoom-in view of the ROIs marked with the yellow dashed box. Intensity bar for Cy3B image indicates the peeling/rupturing percentage of the probes. Scale bar = 10 µm. (c) Microscopy images of NIH3T3 cells incubated on peeling probe or TGT substrates for ~90 min, fixed and stained for nucleus (DAPI), actin (SirActin), and YAP (Alexa488 conjugated antibody). Scale bar = 5 µm. Intensity bar indicates the gray value. (d) Quantitative analysis of the spreading area, %peel or %rupture, and YAP translocation for NIH3T3 cells incubated on three substrates for 60 min. For spreading area and tension quantification, data was collected from 3 biological replicates (n = 227, 150, and 105 for cells on peeling probe, 56 pN TGT, and 12 pN TGT substrate). For analysis on YAP translocation, images in DAPI channel were used as masks to quantify the mean fluorescence intensity of nuclear YAP and cytoplasm YAP. Data was acquired from 3 biological replicates (total n = 134, 98, and 86 cells for peeling probe, 56 pN TGT, and 12 pN TGT substrate). Plots show lines at the median and interquartile values. Statistical analysis was performed with one-way ANOVA and Tukey’s multiple comparison test.
(a) Scheme showing that Lat B early treatment inhibits integrin force generation by inhibiting actin polymerization. (b) Representative RICM and Cy3B microscopy images of MEF cells incubated on peeling probe substrate after early Lat B treatment. Cells were treated with 20 µM Lat B after 15 min of plating on the substrate and imaged after 45 min of incubation. Scale bar = 10 µm. (c) Scheme showing that when integrin force signals are already generated on peeling probe substrate, if the force transmission is terminated by late Lat B treatment, with additional BHQ2 peeling strand the peeling probe can rehybridize to the duplex form. (d) Representative RICM and raw Cy3B microscopy images of MEF cells treated with 20 µM Lat B 50 min after seeding. The tension signal remained after 10 min of Lat B treatment and diminished after the addition of excess BHQ2 peeling strand. Scale bar = 5 µm. (e) Quantitative analysis of tension signal changes after Lat B treatment and the addition of BHQ2 peeling strand in n = 30 cells. (f) Linescan of the ROI (yellow dashed line) in (D) before Lat B treatment, with Lat B treatment, and with excess BHQ2 peeling strand.
(a) Scheme showing flow cytometry measurements of the DNA strands uptake in solution. Mouse platelets were incubated with 50 nM of cholesterol peeling strand, TaCT duplex, load-bearing strand, TaCT duplex lacking RGD, and load-bearing strand lacking RGD in Tyrode’s buffer for 30 min and spun down three times to wash away the excess oligos. The association was measured in both Atto647N and Cy3B channels by a flow cytometer. (b) Cy3B and Atto647N median fluorescence intensity (MFI) of platelets incubated with different oligonucleotides. Data collected from 3 replicates (mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple comparison. (c) Scheme showing flow cytometry measurements of the concentration dependent incorporation of cholesterol peeling strand. MEF cells were incubated with 0, 1, 10, and 100 nM of cholesterol peeling strand for 1 h. The excess cholesterol peeling strand was washed away by spinning down in PBS three times, and the fluorescence intensity of cells was measured by a flow cytometer. (d) Representative histogram of cholesterol peeling strand association in cells. Atto647N MFI was plotted from 3 replicates (mean ± SD). Linear relationship between cholesterol strand concentration and cell association was found, R2 = 0.977. (e) Scheme showing the measurement of cholesterol peeling strand dissociation from the cell. NIH3T3 cells were incubated with 100 nM cholesterol peeling strand for 30 min and rinsed with PBS 3 times. Cells were divided into 6 aliquots and the remaining cholesterol strand in cells was measured every 30 min for 150 min by flow cytometry. (f) Representative histogram shows the decay of fluorescence in cells over time. The normalized MFI from 2 replicates was plotted to show the dissociation of cholesterol strand over time in NIH3T3 cells (mean ± SD).
(a) Representative microscopy images of NIH3T3 cells cultured on TaCT substrate, or control substrate lacking cholesterol, lacking RGD, or treated with Lat B (n = 3). Atto647N shows the signal normalized to the background intact probes. Scale bar = 10 µm. (b) Representative flow cytometry histograms show that NIH3T3 cells in TaCT, (−)cholesterol, (−)RGD, and (+)Lat B groups had indistinguishable Cy3B intensity. Quantitative analysis (mean ± SEM) shows that cells cultured on TaCT substrate had similar level of geometric mean fluorescence intensity (gMFI) compared to control groups in Cy3B channel, despite higher Atto647N gMFI. Data collected from 3 replicates for each control, and statistical analysis was performed using ratio paired two-tailed Student’s t-test. (c) Plots show the gMFI of cells after TaCT normalized to different controls in both Atto647N and Cy3B channels (n = 3, mean ± SEM). One sample t-test (two-tailed) was used for statistical analysis to test whether TaCT signal is significantly different in Atto647N and Cy3B compared to a hypothetical value of 1 (representing no TaCT signal in the corresponding control group). TaCT signal in Atto647N channel consistently provides 2 to 3 fold higher gMFI.
(a) Scheme and representative microscopy data showing that TaCT signal is not due to spreading of the cells on the substrate. NIH3T3 cells were incubated on a TaCT substrate doped with a non-fluorescent DNA duplex, or a TaCT substrate lacking RGD doped with a non-fluorescent DNA duplex presenting the RGD motif, and imaged 45 min–60 min after seeding. After confirming the cell spreading, cells were collected, rinsed, and added to non-fluorescent glass substrate to image the cholesterol peeling strand incorporation in the cells. Tension images were normalized to the background of intact probes. Scale bar = 10 µm. (b) Quantitative analysis of the cell spreading area (n = 82 and 74 cells) and cholesterol peeling strand incorporation (n = 58 and 56 cells) for cells incubated on TaCT (+)RGD or (−)RGD substrates. Data was collected from three replicates and statistical analysis was performed using two-tailed Student’s t-test.
(a) Scheme and representative microscopy images showing tension mapping with peeling probe in mouse platelets (n = 3). Tension images were normalized to the background of intact peeling probes, scale bar = 5 µm. (b) Linescan of the ROI (yellow dashed line) shows anti-colocalization of Cy3B and Atto647N intensities from raw data. (c) Scheme and representative images show mouse platelets incubated on the TaCT substrate in different buffer conditions (n = 3). RGD, Ca2+, Mg2+, and ADP leads to platelets activation, and withholding any of them results in no/impaired activation. Tension images were normalized to the background of intact TaCT probes, scale bar = 5 µm.
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Ma, R., Rashid, S.A., Velusamy, A. et al. Molecular mechanocytometry using tension-activated cell tagging. Nat Methods 20, 1666–1671 (2023). https://doi.org/10.1038/s41592-023-02030-7