相間細胞核の力学特性を探査する生物物理学的なアッセイ：基板の歪みのアプリケーションおよびマイクロニードル操作

The overall goal of the following experiments is to measure the mechanical properties of the interface cell nucleus and its physical connection with the surrounding cytoskeleton. This is achieved by applying mechanical strain to cells plated on a transparent silicone membrane and imaging the induced nuclear deformations. Increased nuclear deformations correspond to decreased nuclear stiffness since changes in nucleo, cytoskeletal coupling can also influence the induced nuclear deformations.

Intracellular force transmission between the cytoskeleton and nucleus is directly probed by applying localized strain to the cytoskeleton. The displacement of fluorescently labeled NU nuclear and cytoskeletal structures is then imaged. Results are obtained that display the stiffness of cell nuclei relative to the surrounding cytoskeleton.

Based on the substrate strain experiments, the microneedle manipulation assay reveals that disruption of nucleo cytoskeletal coupling causes defects in intracellular force transmission. The main advantage of these techniques over existing methods like micro pipette aspiration of isolated nuclei, is that they enable the measurements of nuclear mechanics and intact living cells without disturbing normal cellular architecture. These methods can provide insight into how changes in nuclear envelope composition can influence mechanical properties of the nucleus.

They can also be applied to study the role of nuclear mechanics in the context of diseases such as LA neuropathies or cancer. Each strain dish consists of a custom made bottomless plastic dish with a diameter of three inches and a plastic O-ring to hold a silicone membrane, which serves as the cell culture substrate. For preparation of the strain dish clamp a four inch by four inch piece of silicone membrane between an O-ring and the dish carefully cut away the excess membrane, rinse the strain dish with deionized water, and then autoclave the dish before coating the silicone membrane with extracellular matrix molecules mark a reference point on the outside of the membrane in the bottom center.

This landmark will help identify the same cells during the strain experiments for uniaxial strain application. Two parallel stripes of scotch tape are applied around the reference point to restrict the formation of the membrane in one dimension. To provide optimal cell attachment, coat the silicone membranes with three micrograms per milliliter of fibronectin or any suitable extracellular matrix proteins.

Cover the strain dish with an inverted 10 centimeter polystyrene dish and incubate it overnight at four degrees Celsius the following day. Rinse the membrane once with PBS to remove excess protein. Fill the dish with 10 milliliters of growth, medium and set aside.

Once the coated silicone membrane dish has been prepared, trypsin eyes the cells with 0.05%tryin and seed a adherent cells onto the dish. In growth medium. This procedure is demonstrated with mouse embryonic.

Fibroblasts or mes incubate the cells for 24 to 48 hours under normal culture conditions. An inverted microscope equipped for substrate strain experiments includes a strain device consisting of a base plate that fits onto the microscope stage. The plate holds a central cylindrical platin, which serves to apply strain to the central section of the silicone membrane.

The device also has a movable plate that holds the strain dish and can slide up and down on four guidance pins as well as a five pound weight to apply a load. In order to visualize nuclei, incubate the cells in the strain dish with one microgram per milliliter of hooked stain for 15 minutes at 37 degrees Celsius. Following incubation, aspirate off the medium and replace with 15 milliliters of phenol red free growth medium with 25 millimolar heis.

Then screw the dish into the dish holder and carefully apply grease to the perimeter of the bottom of the silicone membrane to ensure gliding of the membrane along the central Platin. Make sure to keep the central section of the membrane clear. Place the base plate onto the microscope stage plate and mount the dish holder carefully on the base plate.

Ensure that in the initial resting position, the silicone membrane of the strain dish loosely rests on the central platin. Next, focus on the bottom of the silicone membrane and find the central black reference dot. The dot will serve as the starting point for all image acquisitions and aids in locating the same cells during and after stretching.

In this image, the central dot fills the entire field of view. Starting from the border of the dot, adjust the focus to visualize the cells and the top of the silicone membrane. Locate well spread cells with centrally located nuclei.

Acquire a face contrast image, which should focus on the cell outline and the silicone membrane. Then acquire a fluorescence image of the nu nuclear hook stain, which should focus on the central plane of the nucleus. After acquiring images of five to 15 cells, move back to the central dot.

Slowly apply the weight to the strain dish resulting in uniform strain application. In the center of the dish. The maximal applied substrate strain is limited by nylon spacers placed on the vertical alignment pins following strain application, focus on the bottom of the silicone membrane and locate the reference dot again, starting from the dot.

Relocate the same cells and again, acquire a phase contrast, end of fluorescence image of each cell in nucleus under full strain, trying to closely match the focal planes of the initial images. This process should not exceed 10 minutes to avoid active remodeling and adaptation of the cell to the strange substrate. After all the corresponding images have been acquired, move the microscope stage back to the starting point.

Carefully remove the weight from the dish holder plate and allow the silicone membrane to relax if necessary. Gently push up the strain dish until it is in the initial position. Then acquire phase contrast and fluorescence.

Images of the post strain cells just as described for the strain images, images of cells and fluorescently labeled nuclei before, during and after strain application are analyzed to compute the normalized nuclear strain. In this demonstration, a custom written MATLAB script is used for the analysis, but several alternative options are available to calculate the applied substrate strain manually match the positions of three to six control points located on the membrane between the corresponding pre full and post strain images. The MATLAB program then computes the applied membrane strain by comparing the positions of matching control points between the pre-train and full strain images.

Positions are also compared for the residual strain between the pre-train and the post strain images. The control points are simultaneously used to register the image pairs, which will help detect damaged or detaching cells. Next, manually select nuclei using a separate MATLAB program that calculates nuclear strain for each individual nucleus.

This is achieved by matching the nuclear size between corresponding pre full and post strain fluorescent images or by matching intra nuclear markers to account for small variations in applied membrane strain between different experiments. The results are expressed as normalized nuclear strain. This is defined as the ratio of the induced nuclear strain to the applied membrane strain computed for each nucleus.

The MATLAB scripts are available from the lambing laboratory upon request. As a final step, each nucleus is validated and measurements from cells that detach or become damaged during strain application are excluded. MEFs spread over two distinct areas on the silicone membrane were imaged with phase contrast and fluorescence microscopy before, during, and after application of 20%UNI axial strain shown here is an example of a successful experiment with valid nuclei from cells that survive the strain application without any damage or detachment.

In contrast, the cells shown here became retracted or partially detached during strain application and are excluded from the analysis. The cell on the left side shows signs of cytoskeletal damage and nuclear collapse, whereas the cell on the right side detaches partly and retracts during strain application. This can be an indication of excessive strain.

Here, an analysis of normalized nuclear strain in a panel of different meth cell lines of Lamin AC deficient mice is shown the cell's ectopically express either an empty vector or wild type lamin A in comparison to myths from wild type litter mates, loss of expression of the nuclear envelope proteins. Lamin AC results in increased normalized nuclear strain. This is indicative of decreased nuclear stiffness, which can be partly or fully restored by reintroduction of wild type Lamin A.To prepare for the microneedle manipulation experiment, incubate a 35 millimeter glass bottom cell culture dish with a low concentration of fibronectin for two hours at 37 degrees Celsius following incubation, wash the dish twice with HBSS and add two milliliters of growth medium to the dish.

Then mouse embryonic fibroblasts are tryps inized with 0.05%trypsin and seeded in growth medium onto the fibronectin coated glass bottom dish in two milliliters of growth medium. To obtain single A adherent non cofluent cells, place the cells back in the incubator overnight to stain the cells, add MIT tracker mitochondrial stain and hooked nuclear stain to the growth medium. Add this growth medium to the cells and follow with incubation.

Next, wash the cells by incubating them in hank's buffered salt saline for five minutes at room temperature. Then add phenol red free growth medium with 25 millimolar. He piece to the cells for imaging.

For the microneedle experiment, insert a previously pulled microneedle into a micro manipulator coupled to an inverted microscope with a digital charge coupled device. Camera and focus the microneedle near a single cell. Acquire one image of the single cell without the microneedle inserted into the cytoskeleton.

In phase contrast, one fluorescent image of the hooks 3 3, 3 4 2 stain and one fluorescent image of the mitochondrial stain with a 60 x objective on an inverted microscope using the micro manipulator, carefully insert the microneedle into the cytoplasm of a cell at a fixed distance away from the nuclear periphery and take one phase contrast image, one fluorescent image of the hooks. 3 3, 3 4, 2 stain and one fluorescent image of the mitochondrial stain. Move the microneedle a specific distance towards the cell periphery at one micrometer per second, using the computer controlled micro manipulator while automatically acquiring images every 10 seconds.

As the microneedle is moving to a specific distance towards the cell periphery, the micro manipulator should be controlled through a computer to achieve consistent micro-manipulation. Procedures finally acquire images after the microneedle is removed from the cytoskeleton images of the fluorescently labeled features of the nucleus and cytoskeleton acquired before, during, and after microneedle manipulation are analyzed to compute the displacement of small intracellular regions. In this demonstration, a custom written MATLAB script is used for analysis, but several other options are available.

The MATLAB script is available from the lambing laboratory upon request to measure the displacement generated by the microneedle. The MATLAB program uses a normalized cross correlation algorithm to compute the shift between the original location of the fluorescently labeled features and their newly identified position. The displacement are then displayed as vector maps and stored as numerical values.

The next step is to use the resulting displacement vector maps to determine the average displacements within the cytoskeleton or nucleus with matlab. First, select three or more points within the cytoskeleton at the strain application site. In the displacement vector map, take the average numerical value to determine the amount of cytoskeletal strain near the application site that was generated during microneedle application.

Next, select three or more points within the nucleus near the strain application site and take the average value. This value represents the amount of nuclear strain near the microneedle application site. Using the same method, determine the nuclear and cytoskeletal strain away from the strain application site from points selected in these respective regions.

A microneedle manipulation assay was performed to measure intracellular force transmission. Shown here are phase contrast images and fluorescence images of a fibroblast labeled with blue nuclear stain and MIT tracker green mitochondrial stain. A microneedle was inserted into the cytoskeleton at a defined distance from the nucleus and subsequently moved towards the cell periphery.

Mitochondrial displacement protract before, during, and after cytoskeletal strain, and then the displacement were plotted as vectors. Each vector represents the displacement computed as the shift between the original location and the newly identified position. Regions with low image intensity or insufficient texture were excluded from the analysis.

The cytoskeletal and nuclear displacement were then quantified in select areas at increasing distances from the strain application site for both control fibroblasts and fibroblasts. With a disrupted nucleo cytoskeletal coupling in cells with intact nucleo cytoskeletal coupling forces are transmitted through the entire cell, resulting in induced nuclear and cytoskeletal deformation that slowly dissipate away from the strain application site. In contrast, cells with disturbed nucleo cytoskeletal coupling display localized displacement near the application site and only little induced deformation further away.

Comparable cytoskeletal strain application at the microneedle insertion site is observed for both control fibroblasts and fibroblasts with a disrupted nucleo cytoskeletal coupling. However, induced nuclear and cytoskeletal displacement at other regions were significantly smaller in the fibroblasts with disrupted nucleo cytoskeleton coupling than in control cells. Thus, a decrease in cytoskeletal and nuclear displacement away from the strain application site indicates that forced transmission between the cytoskeleton and nucleus was disturbed.

After watching this video, you should have a good understanding of how to measure nuclear mechanics and intracellular force transmission between the nucleus ando cytoskeleton. You can use these techniques to study the effect of mutations in nuclear envelope proteins or changes in nuclear envelope composition that occurred during development or in cancer.