This protocol enables the spontaneous self-organization of microtubule tactoids using an anti-parallel cross-linker, MAP65. This system can help us understand microtubule organization and biological systems like mitotic spindles. The main advantage of this technique is that it is a minimalistic system that can recreate spindle-like shapes for microtubules that is highly reproducible and accessible to many labs.
This method is not only applicable to cellular systems, but can also serve as a model for meso-scale liquid crystal studies. To prepare tubulin, first take out an aliquot of unlabeled tubulin containing one milligram of lyophilized tubulin from the minus 80 degree Celsius freezer and keep it on ice. Then, add 200 microliters of cold PEM-80.
To dissolve all the lyophilate, keep the tube on ice for 10 minutes. Next, take out a tube of rhodamine-labeled tubulin containing 20 micrograms of lyophilized tubulin powder from the minus 80 degree Celsius freezer and keep it on ice. Then, add four microliters of cold PEM-80 and keep the tube on ice for 10 minutes to dissolve all the lyophilate.
Once the lyophilized tubulins have dissolved, add 100 microliters of the resuspended unlabeled tubulin solution to the four-microliter rhodamine-labeled tubulin solution. Then, mix the solutions by piping six to seven times very slowly. To store the remaining 100 microliters of unlabeled tubulin solution, first, insert the tube into liquid nitrogen to freeze the solution, then keep the tube at minus 80 degree Celsius for future use.
Next, distribute the tubulin mix into seven new tubes by adding 15 microliters in each. Freeze the tubes in liquid nitrogen as demonstrated previously and store them at minus 80 degrees Celsius for future use. To assemble flow chambers for performing experiments, first, clean a glass slide with double-distilled water and dry it with a lint-free laboratory wipe.
Next, clean the slide first with ethanol followed by double-distilled water. To create a flow path, cut a 40-to 50-millimeter-long piece of double-sided tape. Then, split it in between to create two thinner strips of tape and place the two strips on the slide five to eight millimeters apart.
Next, place silanized cover slips on top of the flow path. Then, seal the slide and cover slipped the double-sided tape strips by gently pressing on the tape region with the back of a pen. If the seal is made well, the tape should turn from translucent to clear.
To remove the extra tape on the edges, cut the tape with a razor blade, leaving only one millimeter from the flow chamber entrance. Then, label the chamber with appropriate experimental parameters For performing the tactoid experiments, first, thaw all the necessary reagents on ice and store them on the ice during work. Next, coat the flow chamber with 20 microliters of 5%non-ionic block copolymer surfactant dissolved in PEM-80 with small drops at both ends of the chamber to prevent the formation of air bubbles inside.
Then, keep the flow chamber in a humid chamber made of Petri dish with a wet, lint-free laboratory wipe for at least five to seven minutes. Next, mix PEM-80, GMPPCPP, Pluronic F-127, dithiothreitol, glucose, polyethylene glycol, the tubulin mix made earlier, and MAP65 with GFP MAP65 for visualization in a sterile tube by pipetting five to six times, keeping the tube on ice. Then, add one microliter of a pre-mixed solution of glucose oxidase and catalase into the tube of tubulin-MAP mixture and mix again by piping seven to eight times.
Divide the total volume of the solution into two portions to be used in separate chambers. Meanwhile, the liquid added earlier to the flow chamber must be removed by capillary action by using a lint-free laboratory wipe at the other end of the chamber, along with the addition of the tubulin-MAP mix to the flow chamber. Once the sample is fully inside the chamber, seal the two ends of the chamber using five-minute epoxy and keep it at 37 degrees Celsius for about 30 minutes to nucleate and grow microtubule tactoids.
For imaging the tactoids by fluorescence microscopy, use objectives with a numerical aperture of 1.2 or more in 60X or higher magnification to collect enough light in fluorescence. Record the images with a complimentary metal oxide semiconductor or a charge-coupled device camera. To maintain the sample, keep it in an environmental chamber set at 37 degrees Celsius.
Alternatively, other stage heaters, including hot air stage heaters and objective temperature-controlled collars with circulating warm water, can be employed for this purpose. As appropriate excitation sources are essential for the correct fluorescence for rhodamine tubulin, use a 561-nanometer laser with at least one milliwatt of power at the sample for good-quality images. However, for GFP MAP65, change the excitation source to a 488-nanometer laser.
If using wide-field epifluorescence microscopy, then use a rhodamine filter cube with an excitation of 540 12.5 nanometers, dichroic of 545 nanometers 12.5 nanometers cutoff, and emission of 575 nanometers long-pass. For GFP MAP, use a GFP filter cube with excitation of 480 15 nanometers, dichroic of 505 nanometers 15 nanometers cut off, and emission of 515 nanometers long-pass. After ensuring that the illumination power and the exposure times are such that the intensity scale for the camera is not saturated, take at least 10 images of different areas to image over 100 tactoids in both the red and green channels, and save them as 16-bit TIFF images for analysis.
Using this protocol, microtubule tactoids whose formation gets completed within 30 minutes can be directly visualized under the microscope. The tactoids are visible with both a 561-nanometer laser in the tubulin channel and a 488-nanometer laser in the MAP65 channel. The images also perfectly overlap with each other.
In this method, the length and width of the tactoids could be measured as well. The intensity profile of a tactoid was found to vary with its width. Moreover, the immobile nature of the microtubule tactoids was demonstrated by fluorescence recovery after photo-bleaching or FRAP experiments, which did not show any recovery of fluorescence after the photo-bleaching.
On the other hand, FRAP experiments revealed a mobile nature of MAP65, for which a gradual recovery and fluorescence could be observed after photo-bleaching. This fluorescence recovery by MAP65 can be fit to a rising exponential decay to find the amplitude and the time scale of recovery. The tactoid experiment part should be completed within 10 to 12 minutes, as the tubulin can go bad on ice quickly, which can be detrimental to the nucleation of tactoids.
Future work with different microtubule cross-linking proteins and cross-linking motor proteins, enzymes that can move microtubules, will continue to expose new information on the self-organization of the mitotic spindle.
