Despite many examples of coordination, most studies examine the actin or microtubule proteins individually. This method permits the visualization of both dynamic polymers in a single reaction. This technique allows the user to assess diverse regulatory proteins for emergent properties that could only be seen with dynamic versions of actin and microtubules.
This technique can be expanded to include lipids or extracts to get closer to building a synthetic cell. Begin by thawing aliquots of PEG-Silane and Biotin-PEG-Silane powders, and dissolving the PEG powders in 80%ethanol to generate the coating stock solutions of 10 milligrams per milliliter mPEG-Silane and two to four milligrams per milliliter Biotin-PEG-Silane. Remove the clean cover slip from ethanol storage using forceps.
Dry with nitrogen gas and store in a clean Petri dish. Coat the cover slips with 100 microliters of coating solution and incubate them at 70 degrees Celsius for at least 18 hours or until use. Cut 12 strips of double-backed double-sided tape to a length of 24 millimeters.
Remove one side of the tape backing and fix pieces of tape adjacent to the six grooves present on a clean imaging chamber. Remove the second piece of tape backing to expose the sticky side of the tape along each chamber groove, and place the chamber tape side up on a clean surface. Mix epoxy resin and hardener solutions one-to-one in a small weigh boat, and use a P1000 tip to place a drop of mixed epoxy between the tape strips at the end of each imaging chamber groove.
Then, place chamber, tape or epoxy side up, on a clean surface. Remove a coated cover slip from 70 degree Celsius incubator and rinse the coated and uncoated surfaces of the cover slips with double distilled water six times. Dry with filtered nitrogen gas and then affix to the imaging chamber with the cover slip coating side toward the tape.
Use a P200 or P1000 pipette tip to apply pressure on the taped glass interface to ensure a good seal between the tape and the cover slip. Incubate the assembled chambers at room temperature for at least five to 10 minutes to allow the epoxy to fully seal the chamber wells before use. Perfusion chambers expire within 12 to 18 hours of assembly.
Use a perfusion pump and sequentially exchange conditioning solutions in the perfusion chamber by flowing 50 microliters of 1%BSA to prime the imaging chamber. Remove the excess buffer from the lure lock fitting reservoir. Then, flow 50 microliters of 0.005 milligrams per milliliter streptavidin and incubate for one to two minutes at room temperature.
Remove the excess buffer from the reservoir. Flow 50 microliters of 1%BSA to block the nonspecific binding and incubate for 10 to 30 seconds. Remove the excess buffer from the reservoir.
Next, flow 50 microliters of warm 1x TIRF buffer and then 50 microliters of stabilized and 50%biotinylated microtubule seeds diluted in 1x TIRF buffer. Set the stage or objective heater device to maintain 35 to 37 degrees Celsius temperature for 30 minutes prior to imaging the first biochemical reaction. Then, set the acquisition interval to every five seconds for 15 to 20 minutes.
Next, set 488 and 647 laser exposures to 50 to 100 milliseconds at 5%to 10%power by first adjusting the polymerization reaction to initiate the actin filament assembly. Acquire the images at 647 nanometers and make appropriate adjustments. Then, adjust the polymerization reaction in a second conditioned perfusion well to initiate the microtubule assembly.
Visualize at 488 nanometers and make appropriate adjustments. Combine three microliters of 10 micromolar 488 tubulin with the 7.2 microliter aliquot of 100 micromolar fluorescently unlabeled tubulin no more than 15 minutes before use. Then, combine three microliters of diluted biotin related actin in appropriate volumes of fluorescently unlabeled and labeled actin such that the final mix will be 12.5 micromolar total actin with 10%to 30%fluorescent label.
Prepare the cytoskeleton mix by combining two microliters of the 12.5 micromolar actin mix stock with the tubulin stock mix no more than 15 minutes prior to imaging. Store on ice until use. Prepare the protein reaction mix by combining all other experimental components and proteins, including 2x TIRF buffer, anti bleach, nucleotides, buffers, and accessory proteins.
Incubate the cytoskeleton mix and the protein reaction mix separately at 37 degrees Celsius for 30 to 60 seconds. To initiate the reaction add the contents of the protein reaction mix to the cytoskeleton mix and mix it. Flow 50 microliters of the reaction mix containing 1x TIRF buffer supplemented with 15 micromolar free tubulin, one millimolar GTP, 0.5 micromolar actin monomers, and appropriate volumes of buffer controls to the perfusion chamber.
Record a time lapse movie using microscope software to acquire every five seconds for 15 to 20 minutes. Condition a new perfusion well and replace the buffer volume with the regulatory protein of interest and buffer controls. Acquire to assess the regulatory proteins for emergent actin microtubule functions.
Example measurements of microtubules and actin filament dynamics are shown in these images. The average time projection of the tubulin channel efficiently visualizes the total microtubule lengths for the line scans used to generate the Kymograph plots. Black dotted lines correspond to the two example Kymographs of the dynamic microtubules.
The growth and disassembly phases of microtubules are shown on each Kymograph. Two example time lapse image montages depicting single actin filaments actively polymerizing are shown here. Elongation rates are calculated as the slope of the plots of the length of actin filaments over time per micromolar actin.
Thus, a correction factor of two must be applied to 0.5 micromolar actin reactions to compare the rates typically determined at the one micromolar actin concentration. Examples from five filaments are shown here. Total internal reflection fluorescence, or TIRF images of the dynamic microtubules in actin filaments polymerizing in the absence or presence of 250 nanomolar Tau are shown here.
Blue dotted lines and arrows mark wear a line was drawn for the line scan plots corresponding to each condition. Overlap between the microtubules in the actin regions can be scored at a set time point per area. Cytoskeletal proteins, specifically actin, microtubules, and their regulators are very sensitive to freeze/thaw cycles.
For consistency and success, use highly pure and properly stored proteins.
