Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.
Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10–60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.
Cells rely on integrins to adhere and exert cellular forces to extracellular matrix. Integrin-mediated cell adhesion and force transmission are crucial for cell spreading1,2, migration3,4, and survival5,6,7. In the long term, integrin biomechanical signaling also influences cell proliferation8,9,10 and differentiation11,12. Researchers have developed various methods to measure and map integrin-transmitted cellular forces at the cell-matrix interface. These methods are based on elastic substratum13, array of micropost14, or atomic force microscopy (AFM)15,16. Elastic substratum and micropost methods rely on the deformation of substrates to report the cellular stress and have limitations in terms of spatial resolution and force sensitivity. AFM has high force sensitivity, but it cannot detect force at multiple spots simultaneously, making it difficult to map cellular force transmitted by integrins.
In recent years, several techniques were developed to study cellular force at the molecular level. A collection of molecular tension sensors based on polyethylene glycol17,18, spider silk peptide19, and DNA20,21,22,23 were developed to visualize and monitor tension transmitted by molecular proteins. Among these techniques, DNA was first adopted as the synthesis material in the tension gauge tether (TGT), a rupturable linker that modulates the upper limit of integrin tensions in live cells22,24. Later, DNA and fluorescence resonance transfer technique were combined to create hairpin DNA-based fluorescent tension sensors first by Chen’s group23 and Salaita’s group20. The hairpin DNA-based tension sensor reports integrin tension in real-time and has been successfully applied to the study of a series of cellular functions21. Afterward, Wang’s lab combined a TGT with the fluorophore-quencher pair to report integrin tension. This sensor is named an ITS25,26. The ITS is based on double-stranded DNA (dsDNA) and has a broader dynamic range (10-60 pN) for integrin tension calibration. In contrast to hairpin DNA-based sensors, the ITS does not report cellular force in real-time but records all historic integrin events as the footprint of cellular force; this signal accumulation process improves the sensitivity for cellular force imaging, making it feasible to image cellular force even with a low-end fluorescence microscope. The synthesis of ITS is relatively more convenient as it is created by hybridizing two single-stranded DNAs (ssDNA).
The ITS is an 18-base-paired dsDNA conjugated with biotin, a fluorophore, a quencher (Black Hole Quencher 2 [BHQ2])27, and a cyclic arginylglycylaspartic acid (RGD) peptide28 as an integrin peptide ligand (Figure 1). The lower strand is conjugated with the fluorophore (Cy3 is used in this manuscript, while other dyes, such as Cy5 or Alexa series, have also been proven feasible in our lab) and the biotin tag, with which the ITS is immobilized on a substrate by biotin-avidin bond. The upper strand is conjugated with the RGD peptide and the Black Hole Quencher, which quenches Cy3 with approximately 98% quenching efficiency26,27. With the protocol presented in this paper, the coating density of the ITS on a substrate is around 1,100/µm2. This is the density we previously calibrated for 18 bp biotinylated dsDNA coated on the neutrAvidin-functionalized substrate by following the same coating protocol29. When cells adhere to the substrate coated with the ITS, integrin binds the ITS through RGD and transmits tension to the ITS. The ITS has a specific tension tolerance (Ttol) which is defined as the tension threshold that mechanically separates the dsDNA of the ITS within 2 s22. ITS rupture by integrin tension leads to the separation of the quencher from the dye that subsequently emits fluorescence. As a result, the invisible integrin tension is converted to a fluorescence signal and the cellular force can be mapped by fluorescence imaging.
To demonstrate the application of the ITS, we use fish keratocyte here, a widely used cell model for cell migration study30,31,32, CHO-K1 cell, a commonly used nonmotile cell line, and NIH 3T3 fibroblast. Coimaging of integrin tension and cell structures is also conducted.
All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC, 8-16-8333-I) of Iowa State University.
1. Synthesis of the integrative tension sensor
2. Preparation of ITS surfaces by immobilizing the ITS on glass-bottomed Petri dishes
NOTE: The reagents used are biotinylated bovine serum albumin (BSA-biotin), avidin protein, and ITS. Chill all reagents and PBS buffer to around 0 °C on ice with an ice bucket.
3. Cell plating onto ITS surfaces
4. Imaging, video recording, and real-time integrin tension mapping
With the ITS, the integrin tension map of fish keratocytes was captured. It shows that a keratocyte migrates and generates integrin tension at two force tracks (Figure 2A). The resolution of the force map was calibrated to be 0.4 µm (Figure 2B). High integrin tension concentrates at the rear margin (Figure 3A). The ITS also shows different specific patterns of different cells. A nonmotile cell, NIH-3T3, forms a specific integrin tension pattern (Figure 3B) quite different from that of the fast migrating keratocyte.
With immunostaining, focal adhesions and the integrin tension of fish keratocytes were coimaged (Figure 4A). The relation between integrin tension and cell structure in keratocytes was studied in detail by Wang et al.25. Integrin tension and stress fibers in CHO-K1 cells were also coimaged (Figure 4B). These experiments indicate that the ITS enables the coimaging of cell adhesion force and cell structures simultaneously under similar imaging settings, thereby facilitating the study of cell structure/force interplay.
Figure 1: Schematics of the ITS. The ITS is a dsDNA decorated with an RGD ligand, a fluorescence quencher, a dye, and a biotin tag. The dye (green-filled circle) is quenched by the quencher. Under integrin tension, the dsDNA is ruptured and Cy3 is freed from quenching and emits fluorescence that can be detected by fluorescent microscopy. Please click here to view a larger version of this figure.
Figure 2: Integrin tension map of a fish keratocyte and the submicron resolution of the ITS. (A) During fast migration, a keratocyte consistently generates an integrin tension map in two force tracks. (B) The distance between the two force tracks is typically 40 µm. The spatial resolution of the cellular force map imaged by the ITS is around 0.4 µm. The linear profile of the Cy3 fluorescent intensity of the area marked by a short red line in the lower left panel is calculated for the calibration of the resolution in cellular force imaging. The result is shown in the lower right panel and fitted with a Gaussian curve. The scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 3: Real-time integrin tension map of a fish keratocyte and NIH-3T3. (A) A fish keratocyte and its integrin tension map. (Upper left) A motile fish keratocyte. (Lower left) Integrin tension map of the keratocyte reported by the ITS. (Upper right) Real-time integrin tension was obtained by frame subtraction. It shows that the newly generated integrin tension is colocalized with the cell rear edge. (Lower right) Integrin tension map (green) and real-time integrin tension (magenta) are presented in one merged figure. The scale bar = 10 µm. (B) An NIH-3T3 fibroblast and its integrin tension map. (Upper left) An NIH-3T3 fibroblast. (Lower left) Integrin tension map of the NIH-3T3 fibroblast. Integrin tension was generated in a stripe pattern. (Upper right) Real-time integrin tension shows that newly generated integrin tension mainly forms at the cell peripheral region. (Lower right) Integrin tension map (green) and real-time integrin tension (magenta) are presented in one merged figure. The scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 4: Coimaging of integrin tension and cell structure. (A) Coimaging of integrin tension, vinculin, and F-actin in a keratocyte. The keratocyte was fixed and immunostained to report vinculin and F-actin. (B) Coimaging of integrin tension, vinculin, and F-actin in a CHO-K1 cell. The scale bar = 10 µm. Please click here to view a larger version of this figure.
The ITS is a highly accessible yet powerful technique for cellular force mapping in terms of both synthesis and application. With all materials ready, the ITS can be synthesized within 1 day. During experiments, only three steps of surface coating are needed prior to cell plating. Recently, we further simplified the coating procedure to one step by directly linking the ITS to bovine serum albumin, which enables direct physical adsorption of the ITS to glass or polystyrene surfaces33. The ITS brings the fluorescent intensity of cellular force signal to a comparable level of cellular structural imaging. The convenient synthesis and sample preparation, modest requirements for microscopy, and high sensitivity for cellular force make the ITS a robust and reliable technique for the study of cell mechanics. In this paper, we presented a comprehensive procedure for ITS synthesis and application.
Conjugating RGD ligand with ssDNA is the most critical procedure in the ITS synthesis. As mentioned in the protocol section, the NHS ester on sulfo-SMCC is not stable in water. The longer it takes to dissolve sulfo-SMCC in water, the more amount of sulfo-SMCC loses NHS ester to hydrolysis and becomes “dud”, which conjugates with DNA through the maleimide-thiol reaction but fails to conjugate with RGD-NH2. As a result, a big portion of DNA molecules will be occupied by the dead sulfo-SMCC and cannot link with RGD, leading to a low yield of ssDNA-RGD. For this reason, dissolving the sulfo-SMCC in water should be completed quickly, generally within 1 min. It is also recommended to check the yield of ssDNA-RGD with electrophoresis. If the yield is higher than 80%, purification is generally unnecessary. Otherwise, DNA purification by polyacrylamide gel is recommended. The recovery rate of DNA by polyacrylamide gel is about 60%–80%.
To synthesize a reasonable amount of RGD-ssDNA-BHQ2, the starting amount of thiol-ssDNA-BHQ2 should be at least 20 nmol (1 mM x 20 µL). Note that DNA modifications significantly reduce the quantity of the ssDNA ordered from DNA companies. For example, if 1,000 nmol of ssDNA with two modifications is ordered, the guaranteed yield may only be 20 nmol. If this is the case, order at least 1,000 nmol of ssDNA for the RGD-ssDNA conjugation. Researchers can also design their own sequences for ITS constructs. Normally, we keep the GC content in the ITS higher than 70% to improve the dsDNA thermal stability. DNA sequence analysis can be performed to minimize the probability of forming self-hairpin, self-hybridization, stable secondary structure, etc. The analysis tool is available online. In the manuscript, the upper strand DNA is the same for ITSs with different Ttol. We vary the biotin location at the lower strand DNA to achieve a different Ttol. However, varying the location of RGD on the upper strand can be also applied to tune the Ttol of the ITS. The calculation of Ttol for a different dsDNA geometry is provided by Wang and Ha22 and Mosayebi et al.34, especially for the dsDNA in the unzipping mode35,36 and the shear mode37,38.
Although ITS surfaces record all integrin tensions that have taken place in the past, by recording a video of the cellular force map consecutively, researchers can compute the integrin tension produced in the latest frame interval by subtracting the previous frame from a current frame to generate the ‘quasi-real-time cellular force map’. The frame interval in the ITS video varies depending on the cell types but is usually 20 s for fast migrating cells and 1–2 min for stationary cells. The frame subtraction method reports the quasi-real-time integrin tension while retaining the high sensitivity for integrin tension imaging due to the signal accumulation effect. Photobleaching is minimal between two consecutive images and, therefore, has no observable influence to the frame subtraction method.
The authors have nothing to disclose.
This work was supported by the startup fund provided by Iowa State University and by the National Institute of General Medical Sciences (R35GM128747).
BSA-biotin | Sigma-Aldrich | A8549 | |
Neutravidin | Thermo Fisher Scientific | 31000 | |
Streptavidin | Thermo Fisher Scientific | 434301 | |
upper strand DNA | Integrated DNA Technologies | N/A | Customer designed. DNA sequence is shown in PROTOCOL section |
lower strand DNA | Integrated DNA Technologies | N/A | Customer designed. DNA sequences are shown in PROTOCOL section. |
sulfo-SMCC | Thermo Fisher Scientific | A39268 | |
Cyclic peptide RGD with an amine group | Peptides International | PCI-3696-PI | |
IMDM | ATCC | 62996227 | |
FBS | ATCC | 302020 | |
Penicillin | gibco | 15140122 | |
TCEP | Sigma-Aldrich | C4706 | |
200 uL petri dish | Cellvis | D29-14-1.5-N | |
NanoDrop 2000 | Thermo Scientific | N/A | spectrometer |
SE410 Tall Air-Cooled Vertical Protein Electrophoresis Unit | Hoefer | SE410-15-1.5 | Device for electroporesis |
CHO-K1 cell line | ATCC | CCL-61 | |
NIH/3T3 cell line | ATCC | CRL-1658 | |
Anti-Vinculin Antibody | EMD Millipore | 90227 | Primary antibody for vinculin immunostaining |
Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 | Invitrogen | A28175 | Secondary antibody for vinculin immunostaining |
Alexa Fluor 647 Phalloidin | Invitrogen | A22287 | |
Eclipse Ti | Nikon | N/A | microscope |