The study of real time cell response to mechanical stimulation is accomplished by first growing cells expressing GFP fusion proteins on glass bottom dishes for 24 hours. The second step is to coat the A FM tip with biotinylated fibronectin. Then the functionalized a FM tip is brought into contact with the cell surface to allow a strong focal adhesion to form around the A FM tip, allowing direct manipulation of the act cytoskeleton through a matrix integrin actin linkage between the fibronectin coated tip and the cell.
The final step is to mechanically stimulate the live cell by controlled upward movement of the A FM tip, such that the vertical force is applied at the point of cell contact without breaking the strong focal adhesion. Ultimately, results show that cells respond to the mechanical stimulation by reinforcing their attachment to the substrate and by inducing actin cytoskeleton reorganization as recorded with real-time fluorescence optical imaging. Hi, I am Andrea Trake, assistant professor in the Department of Systems Biology and Translational Medicine at the Texas a and m Health Science Center.
Hi, I'm Sum Lim. I'm postdoctoral fellow in Department of Systems Biology and Translational Medicine at Texas a and m Health Science Center. Today we'll show you a procedure for studying adaptive cellular remodeling to mechanical stimulation.
We use this procedure in our laboratory to investigate real-time cytoskeletal remodeling in live vascular smooth muscle cells. So let's Get started. For these studies, an inverted Olympus IX 81 microscope with a total internal reflection fluorescence or turf attachment combined with a CSU 22 Yokogawa scanning head is used.
A bio scope SZ atomic force microscope is mounted on top of the inverted optical microscope. The entire system is mounted on a research grade optical table and has been previously described in detail. This integrated microscope uses two quant EM electron multiplying charge coupled device or E-M-C-C-D cameras.
One is for turf imaging and the other for confocal imaging. The confocal assembly, which is composed of the spinning disc scanner external filter wheel, and an E-M-C-C-D camera is mounted on an aluminum base plate placed on top of a silicon pad necessary for vibration damping. The entire confocal assembly connects with the microscope through a flange with a threaded collar.
Thus the assembly can be decoupled from the microscope during atomic force microscopy or a FM measurements to avoid vibration from the spinning disc scanner. An argon krypton laser is used as an excitation source for both turf and confocal imaging modes. The laser can be coupled to either of the two optical fibers, which deliver the laser to the turf attachment or the confocal scanning head.
Two, computers control the system. One utilizes slide book software, which controls the optical imaging and the other nano scope software, which controls the A FM 24 hours prior to the mechanical stimulation experiment placed live cells expressing GFP constructs targeted against specific proteins in a glass bottom cell culture dish. On the day of the experiment, replace the cell culture medium with phenol red free medium.
Because the a FM experiment is very noise sensitive, avoid talking or touching the optical table or microscope during the experiment to minimize noise. In the a FM measurements, decouple the spinning disc confocal from the microscope. The experiment starts by mounting the cell culture dish on the a FM stage with a magnetic collar to fix the dish to the magnetic microscope stage.
The V-shaped cantilever on the A FM probe is customized with the two micron biotinylated glass bead. The cantilever can easily be seen using the microscope. The bead is glued at the very end of the cantilever, and this is where the coating solution should be applied dropwise.
To prepare the a FM probe for the experiment, first mount the tip in the glass holder and then place the glass holder on the table mount to coat the tip. Wash the tip by carefully dripping 10 microliters of docos phosphate buffered saline or DPBS at the very end of the tip, and then use a kim wipe to dry the tip. Repeat this procedure five times after washing with DPBS at a one milligram per milliliter.
Avid in solution. Dropwise over the tip. Allow the tip to incubate for five minutes following avadon coating.
Wash the tip five more times with DPBS. Then cross-link biotinylated fibronectin onto the avadon by adding a one milligram per milliliter solution. Dropwise onto the tip and incubating for five minutes.
Finally, wash the tip again five times with DPBS leaving the last drop on the tip. Once the tip has been prepared, mount the tip on the a FM scanner. After mounting the a FM tip, place a protective silicon skirt around the holder.
Then set up the microscope of video viewing. Next, align the a FM tip in the center of the field of view. Set the optical lever by placing the laser beam at the very end of the cantilever.
Then change the objective to the proper high magnification objective for single cell imaging. Then bring the cell, which will be mechanically stimulated into the field of view and choose a place where the A FM tip should land on the cell surface. Finally, lower the a FM tip until it touches down on that specific cell.
After bringing the tip in contact with the cell, wait for approximately 20 minutes. This allows for a strong focal adhesion to form at the a FM point of contact on the cell surface. Following the waiting period, the mechanical stimulation procedure can begin to prepare for the mechanical stimulation experiment.
Switch from video camera to E-M-C-C-D camera to allow for viewing the fluorescent cells for imaging. Then acquire an initial image of the cell at rest. Use spinning disc and vocal microscopy for visualization of the act in cytoskeleton throughout the cell body.
Conversely, use turf to visualize focal adhesions to stimulate the cell. Use the cantilever to apply a controlled upward movement in discrete steps every three to five minutes. While acquiring the A FM data, it is important that the operator constantly monitor the voltage of the Z piso position to determine the proper timing for the vertical pole.
Simultaneous to a FM data acquisition record a a confocal or turf image of the cell after each vertical pull using the slide book software. Finally withdraw the a FM tip from the cell upon completion of the stimulation experiment. A vascular smooth muscle cell transfect with actin MRFP and vcul.
GFP was imaged using a FM at the apical cell surface. The same cell was imaged using turf at the basal cell surface. Here the two images are overlapped.
An image of the same cell was also acquired using spinning disc confocal throughout the cell body and is shown as a maximum projection of a 3D stack. Good overlaps between the A FM and spinning disc Confocal images are shown. Her vascular smooth muscle cell transfected with actin MRFP was mechanically stimulated by the A FM and imaged by spinning disc confocal microscopy.
The errors indicate acting fibers that show remarkable restructuring upon a FM stimulation. Some act fibers polymerize while others depolymerize due to the mechanical stimulation, other acting fibers bundle together. Due to act restructuring, We've just shown you how to mechanically stimulate a life selling culture using EFM while simultaneously recording CY rearrangement using optical imaging methods.
When doing this procedure, it is important to remember that the a FM experiment is extremely noisy, sensitive. So that's it. Thank you for watching and good luck with your statements.
