This DNA-based integrative tension sensor, or ITS, converting force signal to fluorescence locally enables the direct imaging of cellular force with fluorescence microscopy. ITS has a high spatial resolution and a high sensitivity. The activation threshold is tunable, allowing selective image of integrin tension at different levels.
After receiving the customized single-stranded DNA, or ssDNA, of interest, to conjugate RGD peptide to the thiolated ssDNA quencher, first add 100 microliters of 0.5-molar EDTA in 400 microliters of water to about 450 microliters of freshly prepared TCEP solution. Then, add 10 microliters of the TCEP-EDTA solution to 20 microliters of one-millimolar thiol DNA quencher in PBS for a 30-minute incubation at room temperature. To conjugate RGD amine with sulfo-SMCC, add 40 microliters of freshly prepared 23-millimolar sulfo-SMCC to 100 microliters of freshly prepared 11-millimolar RGD amine solution for a 20-minute incubation at room temperature.
To conjugate RGD amine SMCC with the thiolated ssDNA quencher, mix the entire volume of the thiolated ssDNA quencher solution with the entire volume of the RGD amine SMCC solution for a one-hour incubation at room temperature. At the end of the incubation, transfer the solution to four-degree Celsius storage overnight. The next morning, mix three-molar sodium chloride with the thiol-conjugated DNA solution and minus 20-degree Celsius, chilled, 100%ethanol at a one-to-10-to-25 ratio, and place the reaction at minus 20 degrees Celsius for about 30 minutes.
When the DNA has precipitated, collect the DNA by centrifugation, and resuspend the pellet in PBS for measurement of the DNA concentration on a spectrometer. For integrative tension sensor, or ITS, synthesis, mix the upper and lower strand DNA solutions at a 1.1-to-one-molar ratio, and incubate the reaction overnight at four degrees Celsius before placing aliquots of the mixture at minus 20 degrees Celsius for long-term storage. For ITS immobilization, coat a 29-millimeter-diameter, glass-bottom Petri dish with 200 microliters of 0.1-milligram-per-milliliter biotinylated bovine serum albumin, or BSA-biotin, for 30 minutes at four degrees Celsius.
When the BSA-biotin has physically adsorbed on the glass surface, wash the dish three times with 200 microliters of fresh PBS per wash, followed by incubation with 200 microliters of 50 micrograms per milliliter of avidin protein for 30 minutes at four degrees Celsius. At the end of the incubation, wash the dish three times with PBS as demonstrated, and add 200 microliters of 0.1-micromolar ITS for another 30-minute incubation at four degrees Celsius. At the end of the incubation, wash the dish three times with PBS, leaving the last wash in the dish until the cell plating.
To initiate a fish keratocyte culture, use a flat-tipped tweezer to pluck a piece of scale from a fish, and gently press the scale onto the well of an unmodified glass-bottom Petri dish, with the inner side of the scale contacting the glass. Wait for about 30 seconds for the fish scale to adhere to the glass surface of the dish before covering the scale with one millimeter of complete Iscove's Modified Dulbecco's Medium. Then, incubate the scale in a dark and humid box for six to 48 hours at room temperature.
To plate the keratocytes onto the ITS surface, replace the medium from the keratocyte culture dish with 1X cell detaching solution for a three-minute incubation at room temperature. At the end of the incubation, replace the detaching solution with fresh complete medium, and dissociate the cells with gentle pipetting. Then, adjust the cells to the appropriate concentration for plating, and seed them onto the ITS coated dish for 30 minutes at room temperature before imaging.
For quasi-real-time integrin tension imaging in live cells, transfer the cells to the stage of a fluorescence microscope, and select the 40 or 100 times objective. In the imaging software, set the exposure time to one second and the frame interval of 20 seconds to obtain a video of the ITS images. Then, use an appropriate image analysis software program to subtract a previous frame of the ITS video from a current frame to compute the ITS signal newly produced in the latest frame interval.
The newly produced ITS signal represents the integrin tension generated in the latest frame interval, thereby reporting the cellular force in quasi-real-time manner. Here, integrin tension mapping demonstrates the induction of integrin tension at two force tracks during keratocyte migration. The resolution of the force map could then be calibrated to be 0.4 micrometers.
High integrin tension concentrates at the rear margin in motile fish keratocytes. In nonmotile cells, a specific integrin tension pattern that is quite different from that observed in the fast migrating keratocyte is formed. Focal adhesions and the integrin tension of fish keratocytes can be co-imaged to evaluate the relationship between integrin tension and cell structure in cells of interest.
With ITS, cell adhesion force becomes visible with a resolution and a sensitivity comparable to cell structural imaging. This technique paves the road for the study of a force-structure interplay in cells.
