Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

1. Prepare reagents

1. Prepare buffers and reagents as outlined in Table 1 and Table 2. During the experiment, keep all solutions on ice, unless otherwise noted.

Table 1: List of buffers used in this protocol and their components. See the "Recommended Storage Duration" column for guidance on how far in advance each buffer can be prepared.

Table 2: List of reagents used in this protocol. Included are the recommended storage conditions and concentrations, working concentrations of stock solutions used during the experiment, and final concentration in the imaging chamber. Additional notes are given in the far-right column.

2. Prepare Biotin-PEG slides

NOTE: Prepare imaging chambers as close to the start of an experiment as possible, and not more than 2 weeks in advance.

1. Clean coverslips
   1. Position an equal number of 24 x 60 mm and 18 x 18 mm #1.5 coverslips (Figure 1F) in slide-staining jars and slide-washing racks, respectively (Figure 1A,B). Place the slide-washing racks containing 18 x 18 mm coverslips in a 100 mL beaker.
   2. Rinse all coverslips 5-6 times in ultrapure water (18.2 MΩ-cm resistivity) and remove excess liquid after each rinse with a pipette tip attached to a vacuum tube (Figure 1C).
   3. Fill beakers and slide-staining jars containing the coverslips with ultrapure water, seal with parafilm, and sonicate for 10 min.
   4. Fill two 150 mL beakers with 200-proof ethanol. Using tweezers, dip each coverslip into one beaker filled with ethanol, and then the other.
   5. Using tweezers, transfer coverslips to the slide-drying rack (Figure 1D), dry them under nitrogen gas stream and incubate at 37 °C until completely dry (~15 min).
   6. Place the dried coverslips in a single layer inside the plasma cleaner. Form vacuum seal, and then set the Radio Frequency (RF) level of the plasma cleaner to ~8 MHz.
   7. Once the plasma is generated, leave coverslips in plasma cleaner for 5 min. Switch off the plasma cleaner and release the vacuum slowly.
   8. Once the vacuum seal is released, flip the coverslips over and repeat the plasma cleaning for 5 min for the other side of the coverslips.
   9. Alternative to plasma cleaning: In place of steps 2.1.2-2.1.3, sonicate coverslips in a warm solution of 2% detergent (in ultrapure water) for 10 min. Then, thoroughly wash coverslips with ultrapure water and sonicate in ultrapure water 2-3 times (10 min each). Next, wash in ethanol and dry as in steps 2.1.4-2.1.5. Skip steps 2.1.6-2.1.8.
2. Biotin-PEG treatment
   1. Immediately before use, dissolve 400 µL of 3-Aminopropyltriethoxysilane in 40 mL of acetone. Using tweezers, move plasma-cleaned coverslips into the slide-washing rack and slide-staining jars. Submerge coverslips in 3-Aminopropyltriethoxysilane solution and incubate for 5 min^12,13.
   2. Wash all coverslips 5-6 times with ultrapure water.
   3. Transfer coverslips to the slide-drying rack, dry them under a nitrogen gas stream and incubate at 37 °C until completely dry (~20 min).
   4. Lay the dried coverslips on delicate task wipes and label each coverslip on one corner, e.g., 'p' on each 18 x 18 mm coverslip and 'b' on each 24 x 60 mm coverslip (see Figure 2).
   5. On the day of the experiment, prepare a fresh 0.1 M sodium bicarbonate solution by dissolving 0.84 mg of NaHCO₃ in 10 mL of ultrapure water.
   6. Bring aliquots of mPEG-Succinimidyl Valerate (PEG-SVA) and Biotin-PEG-SVA to room temperature, immediately before use. See notes on Polyethylene Glycol (PEG) aliquot preparation in Table 2.
      NOTE: Work quickly because the hydrolysis half-life of the Succinimidyl Valerate (SVA) moiety is ~30 min.
   7. Add 102 µL of 0.1 M NaHCO₃ to 34 mg of PEG-SVA, spin in a benchtop microcentrifuge at 2,656 x g for 20 s, and then mix by pipetting up and down. Dissolve 3 mg of Biotin-PEG-SVA in 27 µL of 0.1 M NaHCO₃ by pipetting up and down. Adjust dilution volumes according to the exact weight of PEG noted on the tubes (see Table 2).
   8. Prepare 100:1 w/w PEG:biotin-PEG mixture by combining 75 µL of PEG-SVA solution and 2.25 µL of Biotin-PEG-SVA solution for 20 coverslips, 100 µL and 3 µL for 30 coverslips, or 125 µL and 3.75 µL for 40 coverslips.
   9. Construct a hydration chamber by placing wet paper towels beneath the tip rack in the bottom of an empty 10 µL tip box (Figure 1E). This will prevent evaporation of the PEG solutions.
   10. Pipette 6 µL of 100:1 PEG-SVA:biotin-PEG mixture onto the center of one 24 x 60 mm coverslip on the labeled side. Place another 24 x 60 mm coverslip on top of the first coverslip such that the pair forms an X-shape, with the sides labeled 'b' facing each other. Place the pair on an empty tip rack in the hydration chamber and repeat for the remaining 24 x 60 mm coverslips.
   11. Pipette 6 µL of PEG-SVA onto the center of one 18 x 18 mm coverslip on the labeled side. Place another 18 x 18 mm coverslip on top of the first coverslip, with the sides labeled 'p' facing each other. Place the pair on an empty tip rack in the hydration chamber and repeat for the remaining 18 x 18 mm coverslips.
   12. Close the hydration chamber and incubate for 3 h or overnight.
   13. Separate the pairs of coverslips and rinse in ultrapure water.
   14. Dry coverslips with a nitrogen stream and place them in a 37 °C incubator to fully dry.
   15. To construct the imaging chamber, stick three strips of double-sided tape on a 24 x 60 mm coverslip on the side labeled 'b'. To the other side of tape strips, attach an 18 x 18 mm coverslip with its side labeled 'p' facing the larger coverslip. This forms two flow chambers for microscopy experiments, with treated surfaces facing each other (Figure 2 and Figure 1G).

Figure 1: Equipment for coverslip treatment and imaging chamber preparation. (A) slide-staining jars for 24 x 60 mm coverslips, (B) slide-washing racks for 18 x 18 mm coverslips, (C) vacuum set-up, (D) slide-drying rack, (E) hydration chamber, (F) coverslips, (G) imaging chamber, (H) slide holder. Please click here to view a larger version of this figure.

Figure 2: Schematic for preparation of imaging chambers using double-sided tape (gray) and PEG/Biotin-PEG treated coverslips. Created with BioRender.com. Please click here to view a larger version of this figure.

3. Polymerize microtubules

1. Prepare GMPCPP seeds
   NOTE: Prepare GMPCPP seeds in a cold room, keeping all reagents, tips, and tubes at 4 °C. GMPCPP seeds can be prepared in advance and stored at -80 °C for up to 1 year. Place fixed-angle ultracentrifuge rotor, containing 1 mL centrifuge tubes, in ultracentrifuge and set the temperature to 4 °C.
   1. Resuspend lyophilized tubulin (Table 2) to ~10 mg/mL in 1X BRB80, immediately before use.
   2. Mix components of GMPCPP seeds as described in Table 3.
      ​NOTE: Keep all tubulin components on ice as much as possible to minimize the polymerization of soluble tubulin.
   3. Clarify mix in a fixed-angle ultracentrifuge rotor at 352,700 x g for 5 min at 4 °C.
   4. Separate the supernatant into 5 µL aliquots, snap freeze them in liquid nitrogen, and store them at -80 °C.
2. Polymerize seeds on the day of the experiment
   1. Warm 1-2 mL of BRB80-DTT (Table 1) to 37 °C.
   2. Place 5 µL aliquot of GMPCPP seeds (-80 °C) from step 3.1.4 on ice and immediately dissolve in 20 µL of warm BRB80-DTT. Spin at 2,000 x g for 5 s at room temperature and tap to mix.
      NOTE: Initial dilution volume may vary between 13 µL and 21 µL and is empirically determined for each batch of seeds. If seeds fail to polymerize, troubleshoot by supplementing initial dilution buffer (step 3.2.2) with 0.5 µM GMPCPP.
   3. Protect from light and incubate at 37 °C for 30-45 min.
      NOTE: The length of the microtubules depends on the duration of incubation. For short microtubules, the incubation time can be as short as 15 min. For long microtubules, the incubation time can be as long as 2 h. Biotinylated microtubules tend to require longer incubation times than non-biotinylated microtubules.
   4. Place fixed-angle ultracentrifuge rotor, containing 500 µL centrifuge tubes, in ultracentrifuge and pre-warm to 30 °C.
   5. After incubation, add 50 µL of warm BRB80-DTT (step 3.2.1) to the polymerized GMPCPP seeds and transfer the mixture to a 500 µL centrifuge tube. Wash the empty tube that contained the GMPCPP seeds with another 50 µL of warm BRB80-DTT, pipette up and down, and add this buffer to the 500 µL centrifuge tube containing the mixture.
   6. Before spinning, mark the rim of the centrifuge tube to indicate where the pellet will be (the pellet will be too small to see). Spin for 10 min at 244,900 x g at 30 °C¹².
   7. Carefully pipette out the supernatant and discard. Resuspend the pellet in 100 µL of warm BRB80-DTT. Tap to mix.
   8. Spin for 10 min at 244,900 x g at 30 °C, aligning the marking with the rotor to pellet in the same place.
   9. Remove the supernatant and resuspend the pellet in 16 µL of warm BRB80-DTT. Transfer the microtubule solution to a clean 0.6 mL microcentrifuge tube. Protect from light and keep at or above room temperature.
      NOTE: After polymerization, keep microtubules at or above room temperature. If they get cold, they will depolymerize. Incubate at 28 °C for added stability.
3. Check microtubules via TIRF microscopy
   1. Pipette a mixture of 4.5 µL of BRB80-DTT and 1 µL of microtubule solution (step 3.2.9) onto a microscope slide. Cover with an 18 x 18 mm coverslip and seal the edges with either clear nail polish or a 1:1:1 mixture of petrolatum, lanolin, and paraffin (valap sealant), which is solid at room temperature and liquid at 95 °C.
   2. Position the TIRF objective beneath the coverslip (see step 4 for recommended microscope settings) and visualize the newly polymerized microtubules at wavelength appropriate for the fluorescently labeled tubulin in the Bright mix (Table 3), to determine what dilution of microtubules to use in the upcoming experiments.

Table 3: GMPCPP seed mix. Components of GMPCPP microtubule seeds, including volume and order of addition. Prepare 5 µL aliquots and store for up to 1 year at -80 °C.

4. Microscope settings

1. Temperature: Set the microscope temperature to 28 °C to view dynamic microtubules.
2. Filters: Use the best combination of filter cubes and emission filters, depending on the fluorescent channels to be imaged. To visualize 488 nm, 560 nm, and 647 nm wavelengths in the same experiment, use a 405/488/560/647 nm Laser Quad Band Set, coupled with emission filters for the designated wavelengths.
3. Align Lasers: Ensure that the Laser beams used in the experiment are aligned. Determine the Laser intensity for the experiment empirically, such that all fluorescent proteins can be imaged with the highest possible signal-to-noise ratio, but do not undergo significant photobleaching over the time course of the experiment.
4. Objective: Use lens paper to clean a 100x objective with 70% ethanol. Prior to imaging, add a drop of microscope immersion oil to the objective.
5. Set up an imaging sequence
   1. For an experiment with 647 nm fluorophore-labeled biotinylated microtubules, 560 nm fluorophore-labeled non-biotinylated microtubules and soluble tubulin, and GFP-labeled protein of interest, image for 20 min. Image the 560 nm and 488 nm channels every 10 s, and the 647 nm channel every 30 s.
   2. To capture a reference image of bundles before the addition of soluble tubulin and MAPs, set up a sequence with one image each in 560 nm and 647 nm wavelengths.

5. Generate surface-immobilized microtubule bundles

NOTE: For the following steps, flow all solutions into a flow chamber by pipetting into one open side, while placing a filter paper against the other side. Protect the imaging chamber from light to reduce photobleaching of fluorescently labeled proteins. Tape the prepared imaging chamber to a slide holder (Figure 1G,H). Follow the steps in Table 4, which correspond to protocol steps 5.2-6.4.

1. Prepare soluble tubulin mix according to Table 5 and keep it on ice.
   NOTE: Soluble tubulin must always be placed on ice to prevent polymerization. Prepare a fresh soluble tubulin mix approximately every 2 h, or when microtubules are no longer polymerizing.
2. To immobilize microtubules via a biotin-neutravidin-biotin linkage, first flow in Neutravidin (NA) solution until the chamber is filled (~7.5 µL) and incubate for 5 min.
3. Wash with 10 µL of MB-cold.
4. Flow in 7.5 µL of the blocking protein κ-casein (KC) and incubate for 2 min.
5. Wash with 10 µL of MB-warm to prepare the chamber for the introduction of microtubules.
6. Dilute the stock of biotinylated microtubules (according to observations in step 3.3.2) in 1X BRB80-DTT and add 1 µL of this dilution to 9 µL of MB-warm. Flow the mixture into the chamber and incubate for 10 min. Use a higher concentration of microtubules for more bundles.
7. Wash away non-immobilized microtubules with 10 µL of MB-warm.
8. Flow 7.5 µL of warm KC into the chamber and incubate for 2 min.
9. During the incubation, prepare a 2 nM solution of the crosslinker protein PRC1 in warm KC. Flow 10 µL of this solution into the flow chamber and incubate for 5 min.
   NOTE: Recombinant PRC1 is expressed and purified from bacterial cells as previously described¹³.
10. To make bundles, flow 10 µL of non-biotinylated microtubules into the chamber and incubate for 10 min. PRC1 will crosslink the non-biotinylated and immobilized biotinylated microtubules^15,16 (Figure 3).
    NOTE: See step 6.1 for preparing the assay mix during the incubation time.
11. Wash the chamber twice with 10 µL of MB-warm. The attached microtubules are stable for around 20 min from this point.

Table 4: Assay steps. List of reagents added to the imaging chamber, with indication of wash (-) or incubation time.

Table 5: Soluble tubulin mix components. Mix at the start of the experiment and keep on ice.

Figure 3: Schematic of addition of assay components to make and image fluorescently labeled bundles and single microtubules. Biotinylated seeds are shown in blue, non-biotinylated seeds and soluble tubulin in red, PRC1 in black, and protein of interest in cyan. Step numbers in figure correspond to those in Table 4. Panel corresponding to step 9 shows a pre-formed bundle (lower left); step 11 shows a newly formed bundle (upper left). Created with BioRender.com. Please click here to view a larger version of this figure.

6. Image microtubule dynamics

1. During the 10 min incubation time in step 5.10, prepare 10 µL of assay mix containing proteins of interest, soluble tubulin, nucleotides, oxygen scavengers¹⁴, and antioxidants according to Table 6. Keep the mix on ice.
2. Load the prepared imaging chamber, taped to slide holder, on the 100x TIRF objective. Use the 560 nm and 647 nm channels to find a field of view that contains an optimum number and density of single microtubules and bundles.
   ​NOTE: If both biotinylated and non-biotinylated microtubules are labeled with the same fluorophore, line-scan analyses of fluorescence intensities can distinguish between single microtubules and bundles.
3. Once a field of view is identified, take a reference image.
4. Carefully flow in the assay mix without disturbing the imaging chamber.
5. Seal the open ends of the chamber with valap sealant.
6. Start the imaging sequence as described in step 4.5.1.

Table 6: Assay mix components. Mix, flow into imaging chamber, and image microtubule dynamics, within 30 min.