Live-Cell Detection of Relative Intracellular Tension Dynamics Using Non-FRET α-Actinin and α-Catenin Tension Indicators
发布: 2026年10月05日第16卷第19期 DOI: 10.21769/BioProtoc.5826 浏览次数: 39
评审: Elena A. OstrakhovitchManasa VL ChanduriAnonymous reviewer(s)
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Abstract
Molecular tension sensors enable the visualization of forces acting on specific intracellular proteins in living cells. Most established genetically encoded sensors rely on Förster resonance energy transfer (FRET), requiring donor–acceptor imaging and correction for spectral bleed-through, which can complicate their application in heterogeneous tissues. We developed non-FRET α-actinin and α-catenin tension indicators containing a force-responsive tension sensor (TS) module composed of an optimized circularly permuted enhanced green fluorescent protein (cpEGFP) scaffold and an elastic (GPGGA)8 linker. The TS module is incorporated into α-actinin or α-catenin, while a C-terminal mCherry serves as a force-insensitive reference for indicator abundance. This protocol describes indicator expression in cultured cells, validation using the myosin II inhibitor blebbistatin, two-color time-lapse imaging and live-cell super-resolution imaging using SRRF-Stream reconstruction based on super-resolution radial fluctuations (SRRF), and calculation of the green/red fluorescence ratio and normalized relaxation ratio. The protocol also specifies essential quality-control procedures, including imaging under nonsaturating conditions with fixed channel-specific acquisition settings across comparisons, registration of the green and red channels, and the use of appropriate vehicle and negative controls. For SRRF-Stream analysis, the green and red channels are reconstructed separately using identical reconstruction settings before ratio calculation. Representative reconstructed images should be compared with the corresponding conventional images to check for reconstruction artifacts. Optional procedures describe imaging of isolated cardiomyocytes and freshly isolated organs from tension-indicator mice. Because molecular loading is inferred from changes in TS fluorescence normalized to mCherry rather than from energy transfer between two fluorophores, the method avoids FRET-specific bleed-through correction; nevertheless, controlled acquisition and channel registration remain necessary. The indicators report relative changes in molecular loading and are particularly useful for resolving protein-specific and subcellular heterogeneity in tension dynamics.
Key features
• Genetically encoded, FRET-free tension sensor module reports relative changes in forces acting on α-actinin or α-catenin.
• C-terminal mCherry provides an internal reference for indicator abundance and motion-related intensity changes.
• Quantitative ratios are calculated from conventional 16-bit images (0–65,535) using ROIs with signals above local background in both channels and no saturated pixels.
• Validated in MDCKII and NIH3T3 cells and cardiomyocytes from genetically targeted mice, the protocol supports routine confocal time-lapse imaging and optional SRRF-Stream reconstruction.
Keywords: Molecular tension sensor
Mechanobiology
α-actinin
α-catenin
Live-cell imaging
Super-resolution microscopy
Blebbistatin
Cardiomyocyte
Ratiometric fluorescence imaging
Relative molecular loading
Chromatic registration
Graphical overview
Graphical overview of the cultured cell and optional mouse workflows. Cultured cells are transfected on day 0, replated onto a glass-bottom dish on day 1, and subjected to blebbistatin treatment and live-cell imaging on day 2. Conventional low-magnification imaging is used primarily to quantify the blebbistatin response, while SRRF-Stream can be used for high-resolution imaging. Optional mouse workflows include blebbistatin-response SRRF-Stream imaging of isolated cardiomyocytes and direct ex vivo SRRF-Stream imaging of freshly excised heart or liver. Under matched acquisition conditions and with appropriate controls, increased molecular loading is expected to decrease TS fluorescence and the TS/mCherry ratio, whereas relaxation is expected to increase both readouts. TS denotes the tension sensor module of the indicator. Thus, relative mechanical loading is inferred from changes in the TS/mCherry ratio.
Background
Mechanical forces regulate cell migration, tissue morphogenesis, homeostasis, and disease progression. Genetically encoded molecular tension sensors have made it possible to measure load across selected proteins in living cells. Most established sensors place a compliant linker between a donor and an acceptor fluorophore and infer extension from changes in Förster resonance energy transfer (FRET) [1–3]. These sensors are powerful, but FRET efficiency depends on both fluorophore separation and dipole orientation, and quantitative imaging generally requires bleed-through correction and carefully matched optical conditions. These constraints can become limiting in thick, optically heterogeneous tissues [3].
The indicators described here utilize a circularly permuted enhanced green fluorescent protein (cpEGFP) derived from G-CaMP8 and an internal spider-silk-derived (GPGGA)8 elastic linker [4–6]. Force-dependent deformation of the tension sensor (TS) module changes green fluorescence. In the α-actinin indicator, the module is inserted between spectrin repeats 1 and 2 (SR1 and SR2). In the α-catenin indicator, it replaces the central M region and is flanked by the flexible linkers GGSGGGSG and GSGCGS. The α-actinin and α-catenin indicators localize primarily to actin-rich structures and adherens junctions, respectively. Because they report different proteins in distinct molecular environments, their absolute green/red ratios should not be compared directly. In both constructs, C-terminal mCherry reports indicator abundance and serves as a reference channel. Under increasing load, TS fluorescence decreases while mCherry remains comparatively stable; under matched acquisition conditions and with appropriate controls, a higher green/red ratio is consistent with relaxation, whereas a lower ratio is consistent with higher molecular loading [4].
The method provides high spatial resolution without FRET-specific bleed-through correction and can reveal dynamic, protein-specific tension patterns in protrusions, cell margins, sarcomeres, and tissue junctions. Optical-tweezer measurements of single molecules of the purified TS module showed a reversible fluorescence response over approximately 0–6 piconewtons (pN) [4]. This range characterizes the purified TS module in single-molecule measurements but does not provide an in-cell calibration for converting each pixel’s green/red ratio into an absolute force value; higher spatial resolution therefore does not produce an absolute force map. Important limitations remain. Absolute ratios depend on expression level, optical path, detector response, and local environment; comparisons should therefore be made within the same indicator and imaging configuration. High expression levels may perturb endogenous complexes and can produce weak intermolecular FRET under artificially high transient-expression conditions, whereas no detectable intermolecular FRET was observed at physiological expression levels in cardiomyocytes [4]. For cell selection, moderate expression is defined as clearly detectable, nonsaturated signals in both channels with the expected localization and no prominent aggregates. Because TS fluorescence is relatively weak, imaging settings should be optimized to yield detectable, nonsaturated signals and kept constant across comparisons.
For selected high-resolution applications, this protocol uses SRRF-Stream reconstruction based on super-resolution radial fluctuations (SRRF), a computational method that reconstructs super-resolved images from temporal fluorescence fluctuations in a sequence of conventionally acquired images [7]. SRRF-Stream is Andor’s proprietary real-time implementation of the general SRRF algorithm in Fusion software.
Materials and reagents
Biological materials
1. MDCKII cells (European Collection of Authenticated Cell Cultures, catalog number: 00062107)
2. NIH3T3 cells (ATCC, catalog number: CRL-1658)
3. pCMV_aActTS_indicator, non-FRET α-actinin tension indicator plasmid (Addgene, plasmid number: 252385)
4. pCMV_aCatTS_indicator, non-FRET α-catenin tension indicator plasmid (Addgene, plasmid number: 252386)
5. (Optional) α-actinin or α-catenin H11 knock-in tension-indicator mice crossed with Ayu-Cre mice; Slc:ICR background [4]
Material availability: The expression plasmids are distributed through Addgene under its applicable terms. The indicator mouse (Mus musculus) lines will be made available upon reasonable request to the corresponding author, subject to institutional arrangements and execution of a Material Transfer Agreement (MTA).
Reagents
1. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, catalog number: 11965092)
2. Fetal bovine serum (FBS) (Biosera, catalog number: FB-1365/500)
3. Penicillin-streptomycin-L-glutamine, 100× (Thermo Fisher Scientific, catalog number: 10378016)
4. Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, catalog number: 25300054)
5. Dulbecco’s phosphate-buffered saline without calcium and magnesium [D-PBS(−)], 10× (FUJIFILM Wako, catalog number: 048-29805)
6. Ultrapure water, produced using a Milli-Q water purification system and stored in a TANKPE060 reservoir (Merck Millipore)
7. Opti-MEM I reduced serum medium (Gibco, catalog number: 31985070)
8. Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, catalog number: 11668019)
9. Gelatin (Sigma-Aldrich, catalog number: G1890-100G)
10. (−)-Blebbistatin, myosin II inhibitor (FUJIFILM Wako Pure Chemical Corporation, catalog number: 021-17041)
11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)
12. (Optional) Para-aminoblebbistatin, a less phototoxic/nonfluorescent blebbistatin derivative [8] (Cayman Chemical, catalog number: 22699)
13. HEPES buffer solution, 1 M (Dojindo Laboratories, catalog number: 345-06681)
14. (Optional for cardiomyocyte isolation) Heparin (Mochida Pharmaceutical, catalog number: N/A)
15. Hanks’ balanced salt solution (HBSS), 10× (Gibco, catalog number: 14185052)
16. Magnesium sulfate heptahydrate (MgSO4·7H2O) (FUJIFILM Wako, catalog number: 131-00405)
17. Taurine (FUJIFILM Wako, catalog number: 201-00112)
18. D-glucose (Sigma-Aldrich, catalog number: 07-0680)
19. 2,3-Butanedione monoxime (BDM) (Sigma-Aldrich, catalog number: B0753-25G)
20. Calcium chloride dihydrate, nuclease and protease tested (Nacalai Tesque, catalog number: 08895-15)
21. Collagenase type II (Worthington Biochemical, product code: CLS-2, catalog number: LS004176)
22. Bovine serum albumin (BSA) (FUJIFILM Wako, catalog number: 013-27054)
23. Laminin (Sigma-Aldrich, catalog number: L2020-1MG)
Solutions
1. Complete DMEM (see Recipes)
2. Antibiotic-free complete DMEM (see Recipes)
3. Imaging medium (see Recipes)
4. 1× D-PBS(−) (see Recipes)
5. 0.1% gelatin coating solution (see Recipes)
6. Blebbistatin stock (see Recipes)
7. Para-aminoblebbistatin stock (see Recipes)
8. Perfusion buffer (see Recipes)
9. 100 mM calcium chloride stock (see Recipes)
10. Digestion buffer (see Recipes)
11. Dissociation buffer (see Recipes)
12. Laminin coating solution (see Recipes)
Recipes
1. Complete DMEM
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| DMEM | n/a | 445 mL |
| FBS | 10% (v/v) | 50 mL |
| Penicillin-streptomycin-L-glutamine | 1× | 5 mL |
| Total | n/a | 500 mL |
Prepare aseptically. Store at 4 °C.
2. Antibiotic-free complete DMEM
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| DMEM | n/a | 450 mL |
| FBS | 10% (v/v) | 50 mL |
| Total | n/a | 500 mL |
Prepare aseptically. Store at 4 °C.
3. Imaging medium
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Complete DMEM | n/a | 49.5 mL |
| HEPES, 1 M | 10 mM | 500 μL |
| Total | n/a | 50 mL |
Prepare aseptically. Store at 4 °C. Equilibrate the imaging medium to room temperature before use.
4. 1× D-PBS(−)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 10× D-PBS(−) | 1× | 100 mL |
| Ultrapure water | n/a | 900 mL |
| Total | n/a | 1,000 mL |
Dilute 10× D-PBS(−) with ultrapure water and sterilize by autoclaving at 121 °C for 15 min. Cool to room temperature before use.
5. 0.1% gelatin coating solution
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Gelatin | 0.1% (w/v) | 0.5 g |
| Ultrapure water | n/a | To 500 mL |
| Total | n/a | 500 mL |
Dissolve gelatin in ultrapure water and adjust to 500 mL. Autoclave at 121 °C for 15 min, cool to room temperature, and store at room temperature. Discard if turbidity or contamination is observed.
6. Blebbistatin stock
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Blebbistatin | 5 mM | 1 mg |
| DMSO | n/a | 684 μL |
| Total | n/a | 684 μL |
Dissolve blebbistatin in DMSO and aliquot the solution into microcentrifuge tubes. Wrap each tube in aluminum foil and store at -20 °C. Before use, warm an aliquot to 37 °C and mix until any precipitate has redissolved. Limit each aliquot to two freeze/thaw cycles and discard if precipitation persists.
7. Para-aminoblebbistatin stock
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Para-aminoblebbistatin | 12.5 mg/mL (40.67 mM) | 500 μg |
| DMSO | n/a | 40 μL |
| Total | n/a | 40 μL |
Dissolve para-aminoblebbistatin in DMSO, aliquot the solution into microcentrifuge tubes, and store at -20 °C. Thaw and mix an aliquot before use. Avoid repeated freeze/thaw cycles.
8. Perfusion buffer (optional)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| HBSS 10× | 1× | 50 mL |
| MgSO4·7H2O | 0.02% (w/v) | 0.1 g |
| Taurine | 0.06% (w/v) | 0.3 g |
| D-glucose | 0.08% (w/v) | 0.4 g |
| BDM | 0.1% (w/v) | 0.5 g |
| Ultrapure water | n/a | to 500 mL |
| Total | n/a | 500 mL |
Combine 50 mL of 10× HBSS with approximately 400 mL of ultrapure water. Add MgSO4·7H2O, taurine, D-glucose, and BDM sequentially, mixing after each addition until completely dissolved. Adjust the final volume to 500 mL with ultrapure water. Prepare fresh on the day of cardiomyocyte isolation and discard any unused buffer.
9. 100 mM calcium chloride stock (optional)
| Reagent | Final concentration | Quantity or volume |
| Calcium chloride dihydrate | 100 mM | 147.0 mg |
| Ultrapure water | n/a | to 10 mL |
| Total | n/a | 10 mL |
Dissolve 147.0 mg of calcium chloride dihydrate in approximately 8 mL of ultrapure water. Adjust the final volume to 10 mL with ultrapure water. Sterilize the solution using a 0.20-μm syringe filter and store at 4 °C.
10. Digestion buffer (optional)
| Reagent | Final concentration | Quantity or volume |
| Collagenase II | 1 mg/mL | 40 mg |
| 100 mM CaCl2 stock | 100 μM | 40 μL |
| Perfusion buffer | n/a | to 40 mL |
| Total | n/a | 40 mL |
Dissolve collagenase II in the perfusion buffer, add 40 μL of the 100 mM CaCl2 stock, and adjust the final volume to 40 mL with perfusion buffer. Prepare immediately before use and maintain at 37 °C until perfusion.
11. Dissociation buffer (optional)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| BSA | 4% (w/v) | 1.2 g |
| Perfusion buffer | n/a | to 30 mL |
| Total | n/a | 30 mL |
Dissolve BSA in the perfusion buffer and adjust the final volume to 30 mL. Keep at room temperature and use on the day of cardiomyocyte isolation. This BSA-containing buffer is used to terminate collagenase digestion, dissociate the heart tissue, and plate isolated cardiomyocytes.
12. Laminin coating solution (optional)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Laminin (1–2 mg/mL) | 1:100 dilution; 10–20 μg/mL | 50 μL |
| 1× D-PBS(−) | n/a | 4.95 mL |
| Total | n/a | 5 mL |
Dilute the laminin stock (1–2 mg/mL, as specified on the supplier’s label) 1:100 in sterile 1× D-PBS(−) to obtain a working concentration of 10–20 μg/mL. Incubate the glass-bottom dish with the diluted laminin at 37 °C for 1 h.
Laboratory supplies
1. 35-mm glass-bottom dishes with a 14-mm diameter No. 1S glass-bottom area (Matsunami Glass, catalog number: D11131H)
2. 60-mm tissue-culture dishes (Thermo Fisher Scientific, catalog number: 150462)
3. 12-well tissue-culture plates (Thermo Fisher Scientific, catalog number: 150628)
4. 200- and 1,000-μL pipette tips (WATSON and SHS-TIP, respectively, or equivalent)
5. Sterile serological pipettes, 5, 10, and 25 mL (VIOLAMO, Thermo Fisher Scientific, or equivalent)
6. Sterile polypropylene conical centrifuge tubes, 15 and 50 mL (Thermo Scientific Nunc, catalog numbers: 339650 and 339652)
7. 1.5-mL polypropylene microcentrifuge tubes (Eppendorf or equivalent) and aluminum foil for protecting blebbistatin solutions from light
8. Modified 35-mm dish lid with an opening, flexible delivery tubing, three-way stopcock, syringe, and adhesive tape for securing the dish and tubing during blebbistatin delivery
9. (Optional for cardiomyocyte isolation) Blunt-end 18 G cannula, 22 G needle, and 20-mL syringe
10. (Optional for cardiomyocyte isolation) Three-way stopcocks, tubing compatible with the peristaltic pump, rubber stopper, and insulating material
11. (Optional for cardiomyocyte isolation) Rounded-tip glass pipettes with progressively smaller bore diameters
12. (Optional for cardiomyocyte isolation) 4-0 PERMA-HAND silk suture (ETHICON, Johnson & Johnson, catalog number: 1687G)
13. (Optional for cardiomyocyte isolation) NB 40 nylon mesh with a 435-μm opening (NBC Meshtec or equivalent)
14. (Optional for cardiomyocyte isolation) Sterile 25-mm syringe filter with a 0.20-μm mixed cellulose ester (MCE) membrane (ADVANTEC, catalog number: 25AS020AS)
15. (Optional for cardiomyocyte isolation and organ collection) Dissection scissors and forceps
16. (Optional for organ imaging) Clean plastic wrap for immobilizing freshly isolated organs (Saran Wrap or equivalent)
Equipment
1. Precision electronic balance (VIBRA/Shinko Denshi Co., Ltd., model: AJII-3200; readability: 0.01 g)
2. Analytical balance (METTLER TOLEDO, model: ME54T; readability: 0.1 mg)
3. Laboratory autoclave (TOMY SEIKO Co., Ltd., model: LSX-500)
4. Adjustable micropipettes covering 2–1,000 μL (Gilson or equivalent)
5. Electric pipette controller (Pipet-Aid XP, Drummond Scientific, catalog number: 4-000-101)
6. Clean bench (S-1300P, Showa Science Co., Ltd.)
7. Humidified direct-heat CO2 incubator (Thermo Scientific, Forma Model 310), set to 37 °C and 5% CO2
8. Tabletop centrifuge compatible with 15- and 50-mL conical tubes (KUBOTA Corporation, model: 4000)
9. Constant-temperature water bath (NTT-2300 immersion thermostat with SB-9 bath, EYELA/Tokyo Rikakikai Co., Ltd.), set to 37 °C for buffer warming or 45 °C for circulation through the water jacket of the Langendorff reservoir, as specified in the procedure
10. Inverted phase contrast microscope (Olympus, model: CKX31)
11. Bürker–Türk hemocytometer with a 0.1-mm chamber depth (ERMA, Tokyo, Japan)
12. Vortex mixer (Scientific Industries, model: Vortex-Genie 2)
13. Nikon Eclipse Ti microscope with C1 confocal scanner, Perfect Focus System, Plan Apo 20×/0.75 NA and Plan Apo 40×/0.95 NA objectives, 405-, 488-, and 561-nm lasers (for multipoint time-lapse imaging)
14. Olympus FV3000 confocal laser-scanning microscope with UPLSAPO40X2/0.95 NA, PLAPON60XOSC2/1.4 NA oil, and UPLSAPO100XO/1.4 NA oil objectives (for standard confocal imaging)
15. Dragonfly 500 spinning-disk confocal system (Andor Technology/Oxford Instruments) on an Olympus IX83 base with Zyla 4.2 Plus sCMOS and iXon Life 888 EMCCD cameras, UPLXAPO20X/0.4 NA, UPLXAPO60XO/1.42 NA oil, UPLSAPO60XS2/1.30 NA silicone-immersion objective, and UPLXAPO100XO/1.45 NA oil objectives (for dual-color, SRRF-Stream, and direct organ imaging)
16. SIL300CS-30CC silicone immersion oil (Evident, catalog number: N4190800)
17. (Optional for cardiomyocyte isolation) Water-jacketed glass reservoir containing a coiled glass perfusate tube, with an approximately 20-mL buffer capacity (manufacturer and model unknown)
18. (Optional for cardiomyocyte isolation) Peristaltic tube pump (EYELA/Tokyo Rikakikai Co., Ltd., model: SMP-23) for recirculation of digestion buffer during Langendorff perfusion
Software and datasets
1. Fiji (ImageJ2 version 2.16.0; ImageJ1 version 1.54p; free and open source) for image processing, region-of-interest selection, fluorescence quantification, and ratiometric image generation [9]
2. Chromagnon (free and open source) for chromatic-shift correction of two-color images [10]
3. Fusion software with SRRF-Stream functionality (Andor Technology/Oxford Instruments; proprietary software supplied with the Dragonfly system) for Dragonfly image acquisition and SRRF-Stream reconstruction
4. EZ-C1 (Nikon; proprietary software supplied with the Nikon C1 system) for Nikon C1 acquisition
5. FLUOVIEW FV3000 (Olympus; proprietary software supplied with the FV3000 system) for FV3000 acquisition
6. Imaris 10.2 (Oxford Instruments; commercial license required; optional) was used only to generate the representative 3D reconstruction and is not part of the core workflow. It is not required for image preparation, ROI measurement, ratio analysis, or statistical analysis
7. Python 3.11.12 with pandas 2.3.3 and NumPy 1.26.4, was used to organize measurements exported from Fiji; SciPy 1.15.2 was uesd for statistical testing; and Matplotlib 3.10.3 and Seaborn 0.13.2 was uesd for plot and heatmap generation, as described in the quantitative-analysis section. Fiji was used for image preparation and ROI measurement. Equivalent statistical and plotting software may be substituted
8. Published datasets and representative movies associated with Fujiwara et al. [4] are available in Supplementary Information: numerical source data (Supplementary Data 1), plasmid DNA sequences (Supplementary Data 2), and time-lapse movies of cultured cells (Supplementary Movies 1–4) and isolated cardiomyocytes (Supplementary Movies 5 and 6)
Note: The versions of the instrument-control software varied between experiments because the acquisition workstations were updated during the study. The protocol does not depend on a specific software version.
Procedure
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文章信息
稿件历史记录
提交日期: Jul 27, 2026
接收日期: Aug 24, 2026
在线发布日期: Sep 8, 2026
出版日期: Oct 5, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
如何引用
Hirai, M. and Fujiwara, K. (2026). Live-Cell Detection of Relative Intracellular Tension Dynamics Using Non-FRET α-Actinin and α-Catenin Tension Indicators. Bio-protocol 16(19): e5826. DOI: 10.21769/BioProtoc.5826.
Download Citation in RIS Format
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