ProtocolMechanotransduction

Application of Fluorescence Resonance Energy Transfer and Magnetic Twisting Cytometry to Quantify Mechanochemical Signaling Activities in a Living Cell

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Science Signaling  26 Aug 2008:
Vol. 1, Issue 34, pp. pl1
DOI: 10.1126/scisignal.134pl1

Abstract

Mechanotransduction is the process by which living cells sense mechanical forces and then convert them into biochemical signaling. Recently, we showed that mechanical stress is transduced from the cell surface to remote cytoplasmic sites within 0.3 seconds, which is at least 40 to 50 times faster than soluble factor–induced signal transduction, and the sites of mechanotransduction colocalize with mechanical stress–induced microtubule displacements. These results suggest that mechanotransduction employs mechanisms different from those of soluble factor–induced signal transduction. Here, we describe a protocol that utilizes fluorescence resonance energy transfer (FRET) and a magnetic twisting cytometry (MTC) device to capture rapid mechanochemical signaling activities in living cells.

Introduction

Mechanotransduction is the process by which living cells sense mechanical forces, and adhesive contacts then convert them into biochemical signals that elicit physiological or pathological responses in tissues, organs, or throughout the entire organism. Several mechanotransduction mechanisms have been proposed (14), the most straightforward of which is that mechanochemical signaling is induced locally at the force-membrane interface by conformational changes of membrane-bound proteins or their substrates and then transduced deeper into the cytoplasm by a cascade of passive diffusion– or active translocation–based biochemical signaling components. The methods commonly used to elucidate the mechanisms of mechanotransduction include immunoblotting or immunostaining (5). However, these approaches are not sensitive enough to detect localized, rapid, transient signaling activities and, thus, have limited spatial and temporal resolution. To overcome these limitations, we employed a FRET (fluorescence resonance energy transfer) technique.

FRET-based reporters are available for a wide variety of proteins, including those involved in cell adhesion, migration, and mechanotransduction, such as the tyrosine kinase Src (3), the guanosine triphosphatases Rho and Rac (68), and focal adhesion kinase (FAK) (9). Most Src molecules are localized to the endosomal membrane, which is attached to the cytoskeleton by microtubule motor proteins, although there is some Src at the plasma membrane (1012). In this Protocol, we use a cytosolic reporter of Src activity that consists of cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), an SH2 domain derived from Src, and a substrate domain derived from the c-Src substrate p130Cas (3, 4). When Src is inactive, the CFP and YFP moieties of the reporter are close to one another (<10 nm), and most emissions from CFP transfer to YFP; thus, FRET occurs and yellow light is emitted. When Src is activated, it phosphorylates the substrate domain of the Src reporter, and this phosphorylated substrate binds to the reporter’s SH2 domain, which leads to a conformational change of the reporter protein. This conformational change in the Src reporter separates the CFP and YFP moieties, which decreases FRET and causes cyan light to be emitted. Therefore, the relative intensity changes of CFP and YFP in images of cells transfected with this Src reporter can be used to measure Src activity in the cell. To visualize enzymatic activity of Src located deep within the cells on the cytoplasmic face of the endosomal membranes, rather than at the plasma membrane, we chose focal planes at ~1 to 1.5 μm above the basal surface of the cell throughout our experiments.

In this Protocol, we describe the use of magnetic twisting cytometry (MTC), a technique that has been widely used to study the mechanical behavior of cells (1316), to apply mechanical stress to cells. Ferromagnetic beads coated with an RGD (Arg-Gly-Asp) tripeptide are attached to cell surfaces, and a magnetic field is used to move the bead and to deform the cell membrane. The amount of stress applied was of a magnitude sufficient to cause localized deformations of the cytoskeleton without affecting the whole cell. The MTC device consists of seven major components: (i) a high-voltage generator to provide the current to magnetize the beads; (ii) one [for one-dimensional (1D) MTC] or three separate (for 3D MTC) bipolar current sources for twisting the beads; (iii) a computer for controlling the twisting apparatus; (iv) an inverted microscope for observing the sample; (v) a charge-coupled device (CCD) camera that uses software capable of synchronizing image capture with step function or oscillatory wave magnetic fields; (vi) a device to maintain the correct temperature of the cultured cells; and (vii) a microscope insert that holds the sample and contains either two pairs of coils (for 1D MTC) or three pairs of coils (for 3D MTC) that generate the alternating electric fields used to magnetize and twist the beads (available commercially from Eberhard). With the FRET technique and MTC, we showed, in contrast to the aforementioned proposed mechanotransduction mechanisms that require diffusion or translocation of biochemical signaling molecules, that mechanical force at the membrane can be transmitted through the cytoskeleton to remote cytoplasmic sites (14, 17) by conformational changes of proteins that are physically linked to the cytoskeleton (16). This mechanical stress–induced signal transduction into the deep cytoplasm occurs too rapidly (< 0.3 s) to be explained by a diffusion- or translocation-based mechanism.

This Protocol outlines the use of MTC and a FRET-based reporter of Src activity to visualize stress-induced Src activity inside living cells with high spatial and temporal resolution (Fig. 1). We also describe the use of a synchronous detection method to assay the colocalization of FRET with the microtubule marker mCherry-tubulin. With the exception of a few minor differences, which we have noted in the Instructions, the procedures for performing the two methods are the same. Performing colocalization experiments requires additional image-collection steps in the MTC and FRET Microscopy portion of the Protocol and an additional image analysis procedure described in the Image Analysis for mCherry-Tubulin (the Synchronous Detection Method) section. Both methods require familiarity with Matlab.

Fig. 1.

Schematic of the Protocol. Live adherent cells are transfected with the CFP-YFP Src reporter or cotransfected with the Src reporter and mCherry-tubulin by lipofection. Twenty-four to 48 hours after transfection, an RGD-coated ferromagnetic bead is attached to the apical surface of the cell. The dish containing the cells is mounted on an inverted microscope so that Src activity in response to local mechanical force induced by twisting the magnetic bead can be observed. For colocalization analysis, a synchronous detection method can be used to visualize displacements of microtubules marked with mCherry-tubulin in the same cell.

Materials

Acetic acid (CH3CO2H)

Calcium chloride (CaCl2)

CFP-YFP cytosolic Src reporter (from Y. Wang, University of Illinois, Urbana, IL)

CO2-independent medium (Invitrogen)

Collagen, type I from calf skin (powder, Sigma, #C9791)

Ferromagnetic beads (Fe3O4; 4.5-μm diameter) (from W. Moller, Gauting, Germany or J. Fredberg, Boston, MA; magnetic beads with various surface properties are commercially available in an assortment of sizes from Spherotech, Inc., Lake Forest, IL)

Dimethyl sulfoxide, sterile-filtered (DMSO; Sigma)

Fetal bovine serum (FBS; HyClone)

Glass-bottomed culture dishes (35-mm diameter; MatTek)

Hanks’ balanced salt solution with calcium and magnesium (HBSS; Invitrogen)

Hepes buffer (1 M, HyClone)

Human airway smooth muscle cells (HASM cells) (from R. Panettieri, University of Pennsylvania, Philadelphia, PA)

Insulin from bovine pancreas (Sigma)

l-Glutamine (100×) (Invitrogen)

Lipofectamine LTX (Invitrogen)

Lipofectamine PLUS (Invitrogen)

mCherry-tubulin expression construct (from R. Tsien, University of California, San Diego, CA)

Nutrient mixture F-12 HAM (Sigma)

Opti-MEM I medium (Invitrogen)

Penicillin-streptomycin (Invitrogen or Sigma)

Phosphate-buffered saline (PBS; HyClone)

Sodium bicarbonate (NaHCO3)

Sodium carbonate (Na2CO3)

Sodium hydroxide (NaOH)

Synthetic RGD-containing peptide [Ac-G(dR)GDSPASSKGGGGS(dR)LLLLLL(dR)-NH2, Peptide-2000 (Telios) (18); Peptides International, Inc., Louisville, KY]

Transferrin (apo-) human (Sigma)

Equipment

Adobe Photoshop

40× 0.55 numerical aperture (N.A.) air and 63× 1.32 N.A. oil-immersion objectives (Leica)

CCD camera (Hamamatsu; model C4742-95-12ERG)

CFP/YFP Dual EX/EM (FRET) Filter sets for FRET experiments (Optical Insights): CFP: excitation S430/25, emission S470/30; YFP: excitation S500/20, emission S535/30. The emission filter set uses a 505-nm dichroic mirror.

Dual-View imaging system (Optical Insights)

Inverted microscope (Leica)

Matlab (Mathworks; Version 7.2)

MTC device (Commercially available via special order from EOL Eberhard, Obervil, Switzerland)

Recipes

Recipe 1: Magnetic Beads
Beads arrive as powder. Resuspend the beads in 95% ethanol to a concentration of 5 mg/ml and store at 4°C.
Recipe 2: Carbonate Buffer
Na2CO3159 mg
NaHCO3293 mg
Distilled water80 ml
Adjust the pH to 9.4 with acetic acid. Add distilled water to a final volume of 100 ml. Sterilize by passing through a 0.2-μm filter, and store at 4°C.
Recipe 3: RGD solution (5 mg/ml)
RGD0.5 mg
DMSO100 μl
Mix well in a sterile hood and store at 4°C.
Recipe 4: Culture Medium for HASM cells
Nutrient mixture F-12 HAM medium (Sigma) supplemented with
FBS10% (v/v)
Penicillin100 U/ml
Streptomycin100 μg/ml
l-Glutamine2 mM
NaOH, sterile-filtered12 mM
CaCl2, sterile-filtered1.7 μM
Hepes25 mM
Mix well in a sterile hood and store at 4°C.
Recipe 5: Collagen solution (3 mg/ml)
Collagen3 mg
Acetic acid20 mM
Distilled water1 ml
Mix well. Sterilize by passing through a 0.2-μm filter and store at 4°C. Use this stock solution to make a working solution of 20 μg/ml in PBS just before use.
Recipe 6: Transfection Medium for HASM cells
Nutrient mixture F-12 HAM medium (Sigma) supplemented with
l-Glutamine2 mM
NaOH, sterile-filtered9 mM
CaCl2, sterile-filtered1.6 μM
Hepes25 mM
Mix well in a sterile hood and store at 4°C.
Recipe 7: Hepes-buffered HBSS
HBSS supplemented with
Hepes20 mM
d-Glucose, anhydrous2 g/l
Sterilize by passing through a 0.2-μm filter and store at 4°C.

Instructions

Coating Magnetic Beads with RGD

Steps 1 to 3 are performed in a sterile hood.

1. Transfer 200 μl of magnetic beads (Recipe 1) into a standard 1.5-ml microfuge tube.

2. Add 1.5 ml PBS to beads, and centrifuge at 1200 rpm for 1.5 min in a microfuge. Discard the supernatant.

3. Add 1 ml Carbonate Buffer (Recipe 2) and 10 μl RGD peptide solution (Recipe 3). The final concentration of RGD will be 50 μg/ml, which is enough to saturate the beads (~1 RGD-peptide per 3 nm2 bead surface area).

4. Mix overnight on a rotator or nutator at 4°C. The beads may be used immediately or stored at 4°C for up to 2 weeks before use.

Cell Culture and Transfections

HASM cells should be maintained in the medium described in Recipe 4 per instructions described in Hu et al. (14). Passages three to eight should be used for these experiments. For lipotransfection, we use the manufacturer’s protocol (Invitrogen).

1. Two days before transfection, add 200 μl type I collagen solution (20 μg/ml, Recipe 5) to the glass surface at the center of the 35-mm glass-bottomed culture dish and allow the collagen to be adsorbed onto the surface overnight at 4°C.

2. One day before transfection, remove the excess collagen solution from the glass portion of the dish and wash the surface three times with 2 ml PBS.

3. Transfer ~300,000 HASM cells into the collagen-coated glass-bottomed culture dish with transfection medium (Recipe 6). The cells should be 60 to 80% confluent at the time of transfection (~50,000 cells on the 14-mm glass bottom)

4. On the day of transfection, dilute 1 μg of the CFP-YFP cytosolic Src reporter plasmid in 200 μl Opti-MEM I medium in a 1.5-ml microfuge tube and mix thoroughly.

Note: If you are using the synchronous detection method, add 1 μg each of the CFP-YFP reporter and the mCherry-tubulin expression construct, and increase the volume of Opti-MEM I medium to 300 μl.

5. Add 1 μl Lipofectamine PLUS to the diluted DNA solution, mix gently, and incubate for 5 min at room temperature.

Note: If you are using the synchronous detection method, increase the volume of Lipofectamine PLUS to 2 μl.

6. Add 2.5 μl Lipofectamine LTX to the diluted DNA solution, mix gently, and incubate for 30 min at room temperature.

Note: If you are using the synchronous detection method, increase the volume of Lipofectamine LTX to 5 μl.

7. Add 200 μl Lipofectamine-DNA complex solution to the glass-bottomed dish of HASM cells.

Note: If you are using the synchronous detection method, add 300 μl of DNA solution to the cells.

8. Incubate the cells at 37°C in 0.5% CO2 for 24 to 48 hours prior to imaging.

MTC and FRET Microscopy

Magnetic twisting cytometry (MTC) is a technique used to exert mechanical stresses on living cells by first magnetizing and then rotating ferromagnetic beads that are bound to the surface of cells. Before starting the experiment, it is important to determine the parameters for applying the magnetic field and for collecting the FRET data. The magnetic twisting field can be varied from 0 to 75 Gauss (G) either as a step function (a constant magnetic field) or as a sinusoidal oscillatory wave of variable frequency. Either type of magnetic field can be used to study biochemical activities that follow mechanical stress. An oscillatory field must be applied when assaying colocalization of biochemical activity with microtubule displacement, however. The stress applied to the cell [in pascals (Pa), 1 Pa = 1 piconewton per square micrometer (pN/μm2)] is defined as the ratio of the applied torque (in pN/μm) to six times the bead volume (in μm3). In practice, the applied stress (in Pa) can be converted from the applied twisting magnetic field (in G) by using the bead constant (in Pa/G) times the applied twisting field (in G). The bead constant reflects the magnetic property of the bead and differs for each batch of beads and, thus, must be calibrated. By changing the magnitude of the magnetic twisting field (in G), one can obtain the bead constant (in Pa/G) when the magnetic beads are immersed in a medium with known viscosity [for a description of this procedure, see (13)]. This step is important for converting applied magnetic fields to applied stresses on the cell surface (13). For example, a 50 G step function applies 17.5 Pa of stress to the cell if the bead constant is 0.35 Pa/G (16). A dish of cells bound to beads may only be magnetized once. Because the magnetic field is applied to the entire dish, only one biochemical activity assay may be performed per dish.

1. On the day of the experiment, centrifuge the tube containing the RGD-coated magnetic beads (1 mg/ml in Carbonate buffer) at 1200 rpm for 1.5 min at room temperature. Discard the supernatant.

2. Resuspend the beads in 1 ml PBS and microfuge at 1200 rpm for 1.5 min. Discard the supernatant.

3. Add 1 ml of PBS to the RGD-coated beads to make a solution of 1 mg/ml.

4. Remove the dish of transfected HSAM cells from the incubator. Remove most of the culture medium from dish so that only the cells in the glass region at the center of the dish are still covered.

5. Add 30 to 40 μl of beads (equivalent to 30 to 40 μg) to the cells adhering to the glass portion of the dish.

6. Return the cells to the 37°C, 0.5% CO2 incubator for 15 min to allow integrin clustering and formation of focal adhesions with the beads.

7. Remove the cells from the incubator, and rinse them twice with 2 ml PBS. Avoid disturbing the cells. Add the PBS gently to the side of the dish and rinse cells by gently tilting the dish. Remove the PBS rinse by pipetting from the edge of the dish.

8. Add CO2-independent medium or Hepes-buffered HBSS (Recipe 7) to the cells carefully to avoid disturbing the cells.

9. Place the dish on the stage of the inverted microscope and locate a transfected cell that is attached to a magnetic bead. Use bright-field illumination to locate a cell to which a single magnetic bead is attached; exclude cells that are attached to more than one bead. Use fluorescence illumination to determine whether that cell has the fluorescent reporter(s).

10. Before beginning the experiment, magnetize the bead by applying a single strong magnetic pulse (~1000 G, <0.5 ms). Apply the force only once.

11. Collect bright-field and phase-contrast images of the cell. If you are using the synchronous method, also collect red fluorescence images of the cell.

12. Collect two or three cyan and yellow fluorescence baseline images before applying the mechanical stress. If you are using the synchronous detection method, also collect red fluorescence baseline images. We use the Dual-View imaging system to simultaneously capture both CFP (1344 × 512 pixels) and YFP (1344 × 512 pixels) images on the same screen.

13. Choose parameters for MTC and FRET microscopy. Parameters for a typical experiment are listed below:

  • Applied stress: 17.5 Pa (50 G step function)

  • Exposure time for each image: 80 to 273 ms

  • Duration of imaging: ~3 s or longer

Note: For a synchronous detection experiment, an oscillatory rather than a step-function field should be applied.

14. Collect cyan and yellow fluorescence images for 3 s or longer while applying constant magnetic field (50 G) to the cells. For a synchronous detection experiment, also collect bright-field and red fluorescence images during the oscillatory stress application.

Image Analysis for FRET Reporter

We used Matlab to create a customized program to calculate the CFP/YFP emission ratio of the Src reporter in living cells. For details about how to create custom programs, refer to the Matlab manual. We used Matlab to perform all of the steps described below, but other commercially available programs, such as MetaMorph (Molecular Devices), can be used as well. The image file (1344 × 1024 pixels, if you are using the Hamamatsu C4742-95-12ERG camera) for each time point consists of both CFP and YFP images in side-by-side frames (1344 × 512 pixels each). All procedures in this section are carried out using Matlab with the Image Processing Toolbox (Mathworks). All these steps can be automated in the Matlab. The CFP/YFP emission ratio increases over time due to photobleaching of the acceptor (YFP). Because there is no Src in the nucleus, the intensity values of pixels within the nucleus do not change during mechanical stimulation. Therefore, a nuclear region is used as a reference to determine the bleaching kinetics for each experiment (see step 7 below). Each pair of CFP and images must be processed as described below.

1. Split each image into its constituent CFP and YFP images. Make separate files by cropping each channel’s image from the original composite and saving each as a separate file.

2. Subtract background signals. Choose a part of the CFP image that does not contain any fluorescent cells and measure the average intensity in this region. Subtract this average background intensity from the entire CFP image. Repeat this procedure with the YFP image.

3. Align the background-subtracted CFP and YFP images with one another pixel-by-pixel by maximizing the normalized cross-correlation coefficient of CFP and YFP images. We use Matlab’s "normxcorr2" function to cross-correlate the images.

4. Because the YFP image is brighter than the CFP image and better shows the cell’s contours, use it to make a binary mask. The binary mask will be used to identify the portions of the CFP images that are outside the cell and to set these pixel values to zero. Determine the intensity threshold and generate a binary mask image by using Matlab’s "graythresh" function based on Otsu’s method (19). Set the pixel value inside the cell to one and the pixel value outside the cell to zero.

5. Multiply the background-subtracted CFP image by the binary mask image so that the region outside the cell contours of the CFP image is set to zero.

6. Normalize the aligned CFP/YFP emission ratios to the lower emission ratio and display the CFP/YFP emission ratio image as a linear pseudocolored image. For example, if the range of the CFP/YFP emission ratio of an HASM cell transfected with cytosolic Src reporter is between 0.1 and 0.4, divide the ratio values by the minimum emission ratio, which is 0.1 in this case. The result is a normalized emission ratio, the range of which is between 1 and 4, which can be displayed using Matlab's default "jet" color bar.

Note: Alternatively, a custom color bar (showing color scale of a contour plot) can be made to display the FRET ratios. This is done by inputting the CFP and YFP image file names in custom-made Matlab software that can generate FRET pseudocolor images automatically.

7. To correct for photobleaching, measure the average intensity values in the nucleus at each time point and normalize them to the average intensity values at time zero. Subtract these values from the normalized CFP/YFP emission ratios of the corresponding cell generated in step 5 to obtain corrected final CFP/YFP emission ratio images.

Image Analysis for mCherry-Tubulin (the Synchronous Detection Method)

We recently demonstrated that sites of stress-induced Src activation colocalize with microtubule deformation (16) by simultaneously measuring Src activity and microtubule displacement. A synchronous detection method (20) was coupled with FRET analysis to visualize and quantify both Src activity and microtubule displacement in response to mechanical stress. Setting up and performing the experiment for synchronous detection requires minor modifications, which are noted in the text, to the above Cell Culture and Transfections and MTC and FRET Microscopy sections. Of particular importance are modifications in steps 13 and 14 in the MTC and FRET Microscopy section. Note that an oscillatory rather than a step-function magnetic field should be applied, and be sure to collect bright-field and fluorescence images during each oscillatory stress application 6 to 10 times per cycle. This method detects displacement or deformations of microtubules at a resolution of 4 to 5 nm (14, 20) and correlates them with the force application. As with the FRET image analysis, this image analysis procedure also requires Matlab. All these steps can be automated in Matlab except for step 4, which is done with Adobe Photoshop. For detailed protocols and steps, see (16).

1. One can use the "imread" function to change the image file to matrix form suitable for use in Matlab. To reduce the noise caused by spontaneous movements of the microtubules, average the images taken during the same twisting phase (if you take 10 images per cycle, there are 10 twisting phases) over 10 cycles to generate one complete cycle of 10 averaged images by using Matlab’s "mean" function.

2. To identify the local displacement of an image, divide the fluorescence image into small arrays of 11 × 11 pixels that overlap by five pixels by inputting corresponding column and row values of the matrix.

3. Obtain the displacement field of the microtubules by comparing corresponding arrays at the same location (11 × 11 pixels) between two images taken at different phases during the twisting cycle and by shifting the arrays of the second image by subpixel increments (1/25 pixels) in the Fourier domain until the mean square differences of the pixel intensities between the shifted array and the corresponding array from the first image reach a minimum. This step requires familiarity with the Fourier transform (20).

4. To assay colocalization, open both the Src activity image generated in the Image Analysis for FRET Reporter section and the microtubule displacement image generated in step 3 above for the same cell with Adobe Photoshop. Overlay the two images to determine whether the Src activation sites and microtubule displacement sites overlap.

Troubleshooting

If you turn on the twisting magnetic field but no stress is applied to the cells, determine whether or not the bead is magnetized. A magnetized bead on the dish will move when the twisting field is turned on. If the bead is not magnetized, repeat step 10 from the MTC and FRET Microscopy Instructions.

If a cell rounds up after the mechanical stress is applied, make sure that the cell is firmly adhered to the substrate. Apply a different form of mechanical stress, such as AFM (atomic force microscopy), optical tweezers, a magnetic gradient, parallel plates, or a glass micropipette, for example, and observe the cell. If the cell also rounds up after one of these other forms of mechanical stress is applied, then the cells are not adequately adhered to the substrate. Be careful to ensure that the magnitude of these applied stresses is within the physiological range to avoid stress-induced cell injury or apoptosis, especially when the duration of stress application is long (minutes to hours).

Notes and Remarks

Applying Stress to a Particular Part of a Cell

If one wants to study how different regions of the cell periphery, such as filopodia or lamellipodia, respond to stresses, one must assess the effect of the size of the bead-cell contact area on the actual magnitude of the stress that is applied to the cell. Another concern when applying mechanical stress to a thin cellular projection is the effect of the underlying rigid surface of the dish. If the cell is plated on a dish coated with a very thin layer of extracellular matrix proteins, most of the applied force will be balanced by the rigid substrate just underneath the cell. If one coats the rigid dish with a thick layer of matrix proteins such as type-1 collagen or other flexible substrates, then one needs to determine the effect of the matrix stiffness on cell biological behavior and on the cell’s response to mechanical forces (16) [see a recent review in (21)].

Using Different-Sized Beads

One can use beads of different sizes for these experiments. Beads smaller than 0.5 μm in diameter may be quickly endocytosed, so it is important to visually determine the subcellular location of the bead when using very small beads when the mechanical stress is applied. Using a combination of small beads and markers of specific subcellular structures, it might be possible to apply stresses to specific intracellular membranes such as phagosomes, lysosomes, mitochondria, or the nucleus. Using beads greater than 20 μm in diameter may induce global cellular deformations. Therefore, it would be difficult or impossible to determine local molecular and structural changes in response to the force delivered by a very large bead. To apply the force only to the plasma membrane and to prevent rapid endocytosis of the bead, use a bead greater than 1 μm in diameter.

Density of Ligand on the Bead

Using different concentrations of ligand to coat the beads may alter biochemical activities in the cytoplasm for a given applied stress. We have found that lower coating densities (<5 μg/ml RGD per mg bead) elicit less mechanical response from the cell for the same applied stress (in other words, the cells are less stiff). Presumably, higher ligand concentrations stimulate an increase in the strength or density of focal adhesions and, thus, confer greater sensitivity to mechanical stress.

Multiple Beads and Force Transmission from One Cell to Another

In principle, one could assay biochemical changes in a cell to which more than one bead is attached to determine the potential additive effects of forces. One may also place a bead on the surface of one cell and observe microtubule deformations and biochemical changes transduced to a neighboring cell (which does not have a bead) through adhesive contacts.

Biochemical Activities Other Than Src's

In principle, this method could be applied to assay any biochemical activities in the cytoplasm or in the nucleus, as long as a biosensor or a reporter is delivered inside the cell for reporting the specific biochemical activity.

Simultaneous Image Acquisition

In order to capture rapid mechanochemical signaling in a living cell using a FRET-based reporter, simultaneous acquisition of fluorescence images via separate channels is essential. In this Protocol, therefore, we use a Dual-View system (Optical Insights) to visualize individual fluorescence images (e.g., CFP and YFP images) simultaneously. This requirement of capturing the CFP and YFP images simultaneously does introduce some limitations to the method, however. When using a Dual-View imaging system with a FRET-based CFP-YFP reporter, the fluorescence emission from the sample is split in two in order to capture the CFP and YFP images side by side. Splitting the emission signal thus raises the lower limit of the level of fluorescence that can be detected. To overcome this limitation, one might use a low-light, highly sensitive CCD camera.

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