TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Actin monomers polymerize in a head-to-tail manner to form thin fibers, called microfilaments. Similarly, tubulin dimers polymerize into longer hollow cylindrical microtubules. These cytoskeleton components maintain cell shape and facilitate cellular motility.

To visualize the actin-microtubule coupling dynamics by total internal reflection microscopy, TIRF, take a customized imaging assembly.

The imaging assembly consists of two coverslips: a top m-PEG silane-coated coverslip and a bottom biotin-PEG silane-coated coverslip. The two coverslips are spaced longitudinally with double-sided adhesive tape and sealed at both ends to create a flow channel.

Pipette BSA into the flow channel to prime the chamber. Add streptavidin solution, and incubate. The streptavidin molecules bind to the biotin molecules attached to the bottom coverslip's PEG-silane.

Flow TIRF buffer into the channel. Add a cytoskeleton mix solution containing unlabeled and green fluorophore-labeled tubulin dimers, along with unlabeled, biotin-labeled, and purple fluorophore-tagged actin monomers.

Next, add a reaction solution containing ATP — actin polymerization-promoting agent, and GTP — tubulin polymerization-promoting agent, and actin-microtubule coupling protein. Incubate.

The tubulin dimers bind to the GTP molecules and polymerize, while the actin monomers bind to the ATP molecules and polymerize to form microtubules and microfilaments, respectively.

Place the imaging assembly under a TIRF microscope. Excite the fluorophores with lasers of appropriate wavelengths.

The excitation light reflects internally, and creates a light field that selectively illuminates the fluorophores in a limited region near the glass-water interface, where there are two media with different refractive indexes. In this region, the microfilaments appear purple, and the microtubules appear green.

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 coverslip from 70 degrees Celsius incubator, and rinse the coated and uncoated surfaces of the coverslips with double-distilled water six times. Dry with filtered nitrogen gas, and then affix to the imaging chamber, with the coverslip coating side toward the tape.

Use a P200 or a P1000 pipette tip to apply pressure on the tape-glass interface, to ensure a good seal between the tape and the coverslip. Incubate the assembled chambers at room temperature for at least five to 10 minutes, to allow the epoxy to fully seal the chamber walls 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 Luer-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 biotinylated 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 in 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.