Structural studies of microtubule-interacting proteins by cryo-electron microscopy are crucial to understand cellular mechanisms. Preparing homogeneous microtubules bound to interacting proteins on a cryo-EM grid are crucial for success. This protocol is simple, cheap, and it allows to prepare microtubules with controlled post-translational modifications in sufficient quantity and quality for cryo-electron microscopy.
Microtubules are very temperature-sensitive. It is therefore important to keep the microtubule-containing solutions warm to prevent depolymerization. To begin, culture genetically modified HCT116 cell line in DMEM cell culture medium until six to 12 confluent plates are obtained.
Wash the cells gently with 10 milliliters of PBS to remove any cell culture medium. Detach the cells from the plates by incubating the cells for five minutes at room temperature with three milliliters of ice-cold PBS supplemented with five-millimolar EDTA, and subsequently, using a cell scraper. Collect the cells in a 50-milliliter tube on ice and spin at 250 G for 10 minutes.
Note the volume of the harvested cells with the volumetric scale on the tube. Resuspend the harvested cell pellet in an equal volume of lysis buffer and lyse cells by sonication. After sonication, for SDS-PAGE analysis, add two microliters of lysate in a tube containing 18 microliters of water and five microliters of 5X SDS sample buffer.
Pipette the remaining lysed cells into a centrifuge tube and spin at 100, 000 G for one hour at four degrees Celsius in an ultracentrifuge rotor to clear the lysate. Using a syringe, remove the cleared lysate without disturbing the pellet and floating layer on top. Add two microliters of cleared lysate into a tube containing 18 microliters of water and five microliters of 5X SDS sample buffer.
Carefully rinse the pellet and scoop up a little bit of the pellet by swirling a P-10 pipette tip through the pellet. Then, add 200 microliters of water and 50 microliters of SDS buffer. Add one-millimolar GTP and 20 micromolar paclitaxel into a collected supernatant to polymerize the microtubules and incubate at 37 degrees Celsius for 30 minutes to allow the microtubules to assemble.
Prepare the cushion buffer by adding 600 microliters of glycerol to 400 microliters of lysis buffer and supplement the mixture with 20 micromolar paclitaxel. Prewarm the buffer to 37 degrees Celsius. Add 800 microliters of cushion buffer to an ultracentrifuge tube.
Then, pipette the GTP-paclitaxel-supplemented lysate carefully on top of the cushion buffer. Place the tube in an ultracentrifuge rotor to spin at 100, 000 G at 30 degrees Celsius for 30 minutes. Mark the outward facing edge of the tube to easily recognize where the microtubule pellet should form.
After centrifugation, remove the cushion buffer carefully using a pipette without disturbing the pellet. Carefully add lysis buffer next to the pellet, rotate the tube a few times to remove as much glycerol as possible from the pellet and the walls of the tube, and then aspirate and repeat the washing step three times. Resuspend the washed pellet gently with a cut tip in 50 microliters prewarmed resuspension buffer and place the tube at 37 degrees Celsius.
Add two microliters of resuspended pellet fraction in 18 microliters of water and five microliters of 5X SDS sample buffer. Prepare the plunge freezer device by installing the blotting paper. Set the device temperature to 30 degrees Celsius and humidification to 100%Allow the device to equilibrate for 30 minutes.
Prepare the settings of the plunge freezer for two applications. For the first application, set the force to 10, two seconds blot time, and zero second wait time. For the second application, set the force to 10, 6.5 seconds blot time, and 10 seconds wait time.
Place the grids with their carbon side up into the metal glow discharger vehicle. Glow discharge the cryo-electron microscopy, or EM grids, at 30 milliamperes for 60 seconds. Cool the polystyrene container assembly with liquid nitrogen and prepare liquid ethane in a metal cup by condensing ethane gas into a cold metal cup.
Dilute the microtubule-interacting protein in equal volume with dilution buffer before applying it to the grids to lower the cell concentration. Place the mixer at 37 degrees Celsius. Use a warm metal block in an insulating polystyrene box if there is no heating block near plunge freezing device.
Grab a glow discharged grid with plunge tweezers and click them into the plunge freezer. Position the polystyrene container with liquid ethane in the plunge freezer and run through the prepared program. First, apply 3.5 microliters of microtubule solution to the grid.
Let the plunge freezer blot the grid. Then, immediately apply 3.5 microliters of diluted protein. And lastly, let the plunger blot and plunge freeze the grid in liquid ethane.
Transfer the grids into a grid storage box and store them in a liquid nitrogen Dewar until imaging. The quality and concentration of the extracted microtubules were assessed by Coomassie-stained SDS gel. A nonlinear regression line of the relative BSA quantities, derived from the interpolation of the microtubule band around 50 kilodalton in the P2 lane with the BSA curve, indicates a final concentration of 1.42 milligram per milliliter.
This agrees well with the number measured with a spectrophotometer by two people. The freshly extracted microtubules were used to make cryo-EM samples. The microtubules look intact and abundant on the micrographs.
Microtubule density per micrograph should not be too high to avoid the microtubules crossing over each other. Broken microtubules indicate that the microtubules are depolymerized, or that the concentration of microtubules was too dense. Microtubules bound to MATCAP had rough edges characterized by electron-dense dots on the microtubule surface.
MATCAP-bound and MATCAP-unbound microtubules could also be distinguished in the calculated 2D classes. Making cryo-EM grids with this method can allow to study the structure of microtubules bound to a specific interacting protein. This can lead to a better understanding of the mechanisms in structural cell biology.
