TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

1. Washing the coverslips

NOTE: Wash (24 mm x 60 mm, #1.5) coverslips according to Smith et al., 2013.

1. Arrange coverslips in a plastic slide mailer container.
2. Submerge coverslips sequentially in the following solutions and sonicate for 30-60 min, rinsing with ddH₂O 10 times in between each solution: ddH₂O with one drop of dish soap; 0.1 M KOH. Store coverslips in 100% ethanol for up to 6 months.
   NOTE: Do not touch glass surfaces with ungloved fingers. Use forceps instead.

2. Coating cleaned (24 mm x 60 mm, #1.5) coverslips with mPEG- and biotin-PEG-silane

NOTE: This protocol specifically uses a biotin-streptavidin system to position actin and microtubules within the TIRF imaging plane. Other coatings and systems may be used (e.g., antibodies, poly-L-lysine, NEM myosin, etc.).

1. Thaw aliquots of PEG-silane and biotin-PEG-silane powders.
2. Dissolve PEG powders in 80% ethanol (pH 2.0) to generate coating stock solutions of 10 mg/mL mPEG-silane and 2-4 mg/mL biotin-PEG-silane, just before the use.
   NOTE: PEG powders often appear dissolved but may not be at the microscopic level. Proper resuspension takes ~1-2 min with constant pipetting. Users are encouraged to pipette an additional 10 times following the appearance of powder dissolution.
   CAUTION: Wear gloves to protect skin from concentrated HCl when making 80% ethanol (pH 2.0).
3. Remove the clean (24 mm x 60 mm, #1.5) coverslip from ethanol storage using forceps. Dry with nitrogen gas and store in a clean Petri dish.
4. Coat coverslips with 100 µL of coating solution: a mixture of 2 mg/mL mPEG-silane (MW 2,000) and 0.04 mg/mL biotin-PEG-silane (MW 3,400) in 80% ethanol (pH 2.0).
   NOTE: For sparse coating (recommended) use 2 mg/ mL mPEG-silane and 0.04 mg/mL biotin-PEG-silane. For dense coating use 2 mg/mL mPEG-silane, 4 mg/mL biotin-PEG-silane.
5. Incubate coverslips at 70 °C for at least 18 h or until use.
   NOTE: Coated coverslips degrade if stored at 70 °C for more than 2 weeks.

3. Assembling imaging flow chambers

1. Cut 12 strips of double-backed double-sided tape to a length of 24 mm. Remove one side of the tape backing and fix pieces of tape adjacent to the six grooves present on a clean imaging chamber.
   NOTE: Tape must be flat for proper assembly, otherwise imaging chambers will leak. Carefully remove the tape backing to avoid bumps. Sliding taped chambers on a clean surface to smooth tape-chamber contacts is recommended.
2. Remove the second piece of tape backing to expose the sticky side of the tape along each chamber groove. Place chamber tape side up on a clean surface.
3. Mix epoxy resin and hardener solutions 1:1 (or according to manufacturer's instructions) in a small weigh boat.
4. Use a P1000 tip to place a drop of mixed epoxy between the tape strips at the end of each imaging chamber groove (red arrow; Figure 1A). Place chamber tape/ epoxy side up on a clean surface.
5. Remove a coated coverslip from the 70 °C incubator. Rinse coated and uncoated surfaces of coverslips with ddH₂O six times, dry with filtered nitrogen gas, and then affix to the imaging chamber with the coverslip coating side toward the tape.
6. Use a P200 or P1000 pipette tip to apply pressure on the tape-glass interface to ensure a good seal between the tape and the coverslip.
   NOTE: With a proper seal, double-sided tape becomes translucent. Imaging chambers lacking sufficient tape chamber contacts will leak.
7. Incubate assembled chambers at room temperature for at least 5-10 min to allow the epoxy to fully seal chamber wells before use. Perfusion chambers expire within 12-18 h of assembly.
   NOTE: Depending on tape placement and the thickness of the double-sided tape used, the assembled chamber will have a final volume of 20-50 µL.

4. Conditioning of perfusion chambers

1. Use a perfusion pump (rate set to 500 µL/min) to sequentially exchange conditioning solutions in the perfusion chamber as follows:
   1. Flow 50 µL of 1% BSA to prime the imaging chamber. Remove excess buffer from the Luer-lock fitting reservoir.
   2. Flow 50 µL of 0.005 mg/mL streptavidin. Incubate for 1-2 min at room temperature. Remove excess buffer from the reservoir.
   3. Flow 50 µL of 1% BSA to block nonspecific binding. Incubate for 10-30 s. Remove excess buffer from the reservoir.
   4. Flow 50 µL of warm (37 °C) 1x TIRF buffer (1x BRB80, 50 mM KCl, 10 mM DTT, 40 mM glucose, 0.25% (v/v) methylcellulose (4,000 cp)).
      NOTE: Do not remove the excess buffer from the reservoir. This prevents the chamber from drying out, which can introduce air bubbles into the system.
   5. Optional: Flow 50 µL of stabilized and 50% biotinylated microtubule seeds diluted in 1x TIRF buffer.
      NOTE: The proper dilution must be empirically determined and contain batch-to-batch variability. A dilution yielding 10-30 seeds per field of view works well with this setup.

5. Microscope preparation

NOTE: Biochemical reactions containing dynamic actin filaments and microtubules are visualized/performed using an inverted Total Internal Reflection Fluorescence (TIRF) microscope equipped with 120-150 mW solid-state lasers, a temperature corrected 63x oil immersion TIRF objective, and an EMCCD camera. Proteins in this example are visualized at the following wavelengths: 488 nm (microtubules) and 647 nm (actin).

1. Set the stage/objective heater device to maintain 35-37 °C at least 30 min prior to imaging the first biochemical reaction.
2. Set the image acquisition parameters as follows:
   1. Set acquisition interval to every 5 s for 15-20 min.
   2. Set 488 and 647 laser exposures to 50-100 ms at 5%-10% power. Set the appropriate TIRF angle for the microscope.
      NOTE: Regardless of microscope setup, the simplest way to set the laser power, exposure, and TIRF angle is to make adjustments on images of either polymer alone (see 5.2.2.1 and 5.2.2.2, below). Users are strongly encouraged to use the lowest laser power and exposure settings that still permit detection.
      1. Adjust the polymerization reaction (Figure 1C) to initiate actin filament assembly and acquire images at 647 nm. Make appropriate adjustments.
      2. Adjust the polymerization reaction in a second conditioned perfusion well to initiate microtubule assembly (Figure 1C) and visualize at 488 nm. Make appropriate adjustments.

6. Preparation of protein reaction mixes

1. Prepare a stock solution of fluorescently labeled tubulin.
   1. Determine the concentration of homemade unlabeled tubulin via spectrophotometry at Abs₂₈₀, as follows:
      1. Blank spectrophotometer with 1xBRB80 lacking GTP.
      2. Calculate the concentration of tubulin using the determined extinction coefficient of 115,000 M^‐1 cm^‐1 and the following formula:
         
   2. Resuspend commercially-made lyophilized lysine-labeled 488-tubulin to 10 µM (1 mg/mL; 100% label) with 20 µL of 1x BRB80 lacking GTP.
   3. Thaw a 7.2 µL aliquot of 100 µM unlabeled recycled tubulin on ice.
      NOTE: Recycled tubulin is critical for successful microtubule assembly in vitro because it removes polymerization-incompetent dimers formed in frozen protein stocks.
   4. Combine 3 µL of 10 µM 488-tubulin with the 7.2 µL aliquot of 100 µM unlabeled tubulin, no more than 15 min before use.
2. Prepare the stock solution of fluorescently labeled actin.
   1. For homemade proteins, determine the concentration and percent label of actin via spectrophotometry AbsAbs₂₉₀ and Abs₆₅₀, as follows:
      1. Blank spectrophotometer with G-buffer.
      2. Calculate the concentration of unlabeled actin using the determined extinction coefficient of 25,974 M^‐1 cm^‐1 and the following formula:
         
      3. Calculate the concentration of lysine labeled Alexa-647-actin using the extinction coefficient of unlabeled actin, the fluor correction factor of 0.03, and the following formula:
         
      4. Calculate the percent label of Alexa-647-actin using the determined ε for Alexa-647 of 239,000 M^‐1 cm^‐1, as follows:
         
   2. Thaw one 2 µL aliquot of 3 µM 100% labeled biotin actin (labeled on lysine residues). Dilute 10-fold by adding 18 µL of G-buffer.
   3. Combine 3 µL of diluted biotinylated actin, and appropriate volumes of unlabeled and labeled actin (above) such that the final mix will be 12.5 µM total actin with 10%-30% fluorescent label.
      NOTE: Greater than 30% percent fluorescent actin monomers (final) can compromise imaging resolution as filaments become difficult to discern from the background.
3. Prepare reaction mixes (Figure 1C).
   1. Prepare cytoskeleton mix (Tube A) by combining 2 µL of the 12.5 µM actin mix stock (6.2.3) with the tubulin stock mix (6.1.4), no more than 15 min prior to imaging. Store on ice until use.
   2. Prepare protein reaction mix (Tube B) by combining all other experimental components and proteins, including 2x TIRF buffer, anti-bleach, nucleotides, buffers, and accessory proteins. An example is shown in Figure 1C.
      NOTE: The final dilution results in a 1x TIRF buffer that contains ATP, GTP, and ionic strength within the estimated physiological range.
4. Incubate Tube A and Tube B separately at 37 °C for 30-60 s. To initiate the reaction, mix and add the contents of Tube B to Tube A (below).

7. Image actin and microtubule dynamics

1. Condition perfusion well (Figure 1B; step 4, above).
2. Initiate actin and microtubule assembly simultaneously by adding the contents of Tube B (reaction mix) to Tube A (cytoskeleton mix) (Figure 1C).
3. Flow 50 µL of the reaction containing 1x TIRF buffer supplemented with 15 µM free tubulin, 1 mM GTP, and 0.5 µM actin monomers and appropriate volumes of buffer controls.
4. Record time-lapse movies using microscope software to acquire every 5 s for 15-20 min.
   NOTE: Initiation of actin and microtubule dynamics occurs within 2-5 min (Figure 2). Longer delays indicate problems with temperature control or concentration-related problems of proteins in the reaction mix.
5. Condition a new perfusion well (step 4) and replace buffer volume with regulatory protein(s) of interest (i.e., Tau) and buffer controls (Figure 1C). Acquire as outlined in step 7 (above) to assess regulatory proteins for emergent actin-microtubule functions.