This method can help answer key questions in the nuclear mechanics field. For example, what is the force required to deform the nucleus in a cell? What are the contributions of cellular and nuclear components to nuclear resistance to deformation?
Or, does nuclear coupling with cellular structures vary with cell type? The main advantage of this technique is that it allows us to apply probing force to deform the integrating nucleo-cytoskeleton in a living adherent cell. Though this method has been used to provide insight into the nuclear mechanical properties in NIH-3T3 cells, it can also be applied to any adherent cell type, such as probing nuclear mechanical properties in Hutchinson-Gilford progerial cells.
To begin this procedure, coat a 35 millimeter glass bottom dish with 5 micrograms per milliliter of fibronectin. Then, culture the NIH 3T3 fibroblast cells in DMEM supplemented with 10%donor bovine serum and 1%Penicillin-Streptomycin on the coated dish at 37 degrees Celsius until desired confluency. Immediately prior to the experiment, wash the cells twice with PBS, followed by a single wash with complete growth medium.
Afterward, add three milliliters of complete growth medium to a glass bottom dish. In this procedure, turn on the microinjector. Using an immersion oil dropper, apply a drop of immersion oil on the objective lens.
Then, clamp the dish tightly to the dish holder and load the dish holder onto the stage. Please note that the cells should be maintained at 37 degrees Celsius and 5%carbon dioxide throughout the experiment. Next, adjust the height of the objective to bring the cells into focus.
Move the microscope stage to find a cell of interest. Using the micromanipulator, move the pipette holder to the highest position. Subsequently, load the micropipette with a 0.5 micrometer diameter tip onto the pipette holder.
Then raise the objective focal plane above plane A and the top of the cell to plane B by adjusting the fine control. Next, set the micromanipulator to coarse control position. Slowly bring the micropipette down to plane B by watching for the silhouette of the micropipette until the micropipette comes fully into focus.
Once the micropipette tip is in focus, set the micromanipulator to fine control. Subsequently, lower the objective to the equatorial plane of the cell and lower the micropipette to around 15 micrometers above that. Set the compensation pressure on the microinjector to the desired pressure.
Wait several seconds for the pressure to stabilize. It is important to check if the pipette tip is broken or clogged, otherwise the force measurement would be inaccurate. Insure that the micropipette is not clogged by using the clean setting on the micromanipulator panel and make sure that the air bubbles emerge from the micropipette tip.
Then, insert the micropipette tip into the cell until it lightly touches the nuclear surface. Create a seal between the micropipette tip and the nuclear membrane by disconnecting the pressure supply tube from the microinjection system, thereby opening the end of the micropipette to the atmosphere. This step creates a negative pressure equal to the compensation pressure on the nuclear surface.
Next, open the image collection software. Set up an AVI acquisition for video or ND acquisition for images in the image collection software. Then, toggle to the corresponding fluorescent imaging channel and begin imaging.
Move the micropipette tip away from the body of the cell until the nucleus detaches from the micropipette. This figure shows the forcing of an NIH 3T3 mouse fibroblast nucleus. As the micropipette tip moves to the right, the nucleus deforms and eventually detaches from the micropipette tip.
The length strain of the nucleus is seen to increase with increasing suction force. The front edge of the nucleus forms a nuclear protrusion and the trailing edge is displaced from its original position. The length of the protrusion is much greater than the trailing edge displacement, suggesting a tight integration between the nucleus and the surrounding cytoplasm.
The time scales are short for relaxation of the nuclear front edge and the nuclear back edge. Direct force probe was used to determine the contribution of intranuclear and cytoskeletal structures to the resistance of the nucleus to deformation. Fluorescent images showed the overlay of the nucleus before and after at the indicated condition.
While no significant differences in nuclear deformation or translation were found after F-actin disruption or microtubule disruption, reducing the menten expression through siRNA-based knockdown resulted in significantly greater nuclear translation and deformation. This suggests that the menten intermediate filaments in fibroblasts are the main cytoskeletal element that helped the nucleus resist local force. After watching this video, you should have a good understanding of how to apply a controlled and known force to the nucleus in a living adherent cell.
