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

Summary

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Abstract

Microtubules are polymers of αβ-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Introduction

Microtubules are biopolymers that form structural scaffolds essential for multiple cellular processes, ranging from intracellular transport and organelle positioning to cell division and elongation. To execute these diverse functions, individual microtubules are organized into micron-sized arrays, such as mitotic spindles, ciliary axonemes, neuronal bundles, interphase arrays, and plant cortical arrays. A ubiquitous architectural motif found in these structures is a bundle of microtubules crosslinked along their lengths¹. An intriguing feature of several microtubule-based structures is the coexistence of bundled microtubules and non-crosslinked single microtubules in close spatial proximity. These microtubule subpopulations can display starkly different polymerization dynamics from each other, as needed for their proper function^2,3,4,5. For instance, within the mitotic spindle, stable crosslinked bundles and dynamic single microtubules are present within a micron-scale region at the cell center⁶. Studying how the dynamic properties of coexisting microtubule populations are specified is, therefore, central to understanding the assembly and function of microtubule-based structures.

Microtubules are dynamic polymers that cycle between phases of polymerization and depolymerization, switching between the two phases in events known as catastrophe and rescue⁷. The dynamics of cellular microtubules are regulated by myriad Microtubule Associated Proteins (MAPs) that modulate the rates of microtubule polymerization and depolymerization and the frequencies of catastrophe and rescue events. It is challenging to investigate the activity of MAPs on spatially proximal arrays in cells, owing to the limitations of spatial resolution in light microscopy, especially in regions of high microtubule density. Moreover, the presence of multiple MAPs in the same cellular region hinders interpretations of cell biological studies. In vitro reconstitution assays, performed in conjunction with Total Internal Reflection Fluorescence (TIRF) microscopy, circumvent the challenges of examining mechanisms by which specific subsets of MAPs regulate the dynamics of proximal cellular microtubule arrays. Here, the dynamics of microtubules assembled in vitro are examined in the presence of one or more recombinant MAPs under controlled conditions^8,9,10. However, conventional reconstitution assays are typically performed on single microtubules or on one type of array, precluding the visualization of coexisting populations.

Here, we present in vitro reconstitution assays that enable the simultaneous visualization of two microtubule populations under the same solution conditions¹¹. We describe a method to simultaneously view the collective activity of multiple MAPs on single microtubules and on microtubule bundles crosslinked by the mitotic spindle-associated protein PRC1. The protein PRC1 preferentially binds at the overlap between anti-parallel microtubules, crosslinking them⁹. Briefly, this protocol consists of the following steps: (i) preparation of stock solutions and reagents, (ii) cleaning and surface treatment of coverslips used to create the imaging chamber for microscopy experiments, (iii) preparation of stable microtubule "seeds" from which polymerization is initiated during the experiment, (iv) specification of TIRF microscope settings to visualize microtubule dynamics, (v) immobilization of microtubule seeds and generation of crosslinked microtubule bundles in the imaging chamber, and (vi) visualization of microtubule dynamics in the imaging chamber through TIRF microscopy, upon addition of soluble tubulin, MAPs, and nucleotides. These assays enable the qualitative evaluation and quantitative examination of MAP localization and their effect on the dynamics of two microtubule populations. Additionally, they facilitate the evaluation of synergistic effects of multiple MAPs on these microtubule populations, across a wide range of experimental conditions.

Protocol

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.

Solution	Components		Recommended Storage Duration	Notes
5X BRB80	400 mM K-PIPES, 5 mM MgCl2, 5 mM EGTA, pH 6.8 with KOH, filter sterilize		up to 2 years	Store at 4 °C
1X BRB80	80 mM K-PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8		up to 2 years	Store at 4 °C
BRB80-DTT	1X BRB80, 1 mM DTT		up to 2 days
Assay Buffer	80 mM K-PIPES, 3 mM MgCl2, 1 mM EGTA, pH 6.8, 5% sucrose (OR 1X BRB80, 5% sucrose, 2 mM MgCl2)		up to 1 year	Store at 4 °C
Master Buffer (MB)	Assay Buffer, 5mM TCEP		1 week	Prepare on the day of experiment; Separate into two tubes: MB-warm at room temperature and MB-cold on ice; include 1 mM DTT if using fluorescent dyes
Master Buffer with MethylCellulose (MBMC)	1X BRB80, 0.8% methylcellulose, 5 mM TCEP, 5 mM MgCl2		1 week	Prepare on the day of experiment; include 1 mM DTT if using fluorescent dyes
Protein Dilution Buffer (DB)	MB, 1 mg/mL Bovine Serum Albumin (BSA), 1 µM ATP		1 day, on ice	Prepare on the day of experiment; include 1 mM DTT if using fluorescent dyes
Oxygen Scavenging Mix (OSM)	MB, 389 µg/mL catalase, 4.44 mg/mL glucose oxidase, 15.9 mM 2-mercaptoethanol (BME)		1 day, on ice	Prepare on the day of experiment
Oxygen Scavenging Final (OSF)	MB, 350 µg/mL catalase, 4mg/mL glucose oxidase, 14.3 mM BME, 15 mg/mL glucose		use within 30 min	Prepare immediately before use by adding 1 µL of glucose to 9 µL of OSM

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.

Reagent

Storage Concentration

Storage Solvent

Storage Temperature

Working Concentration

Final Concentration

Recommended Storage Duration

Notes

Neutravidin (NA)

5 mg/mL

1X BRB80

-80°C

0.2 mg/mL

0.2 mg/mL

up to 1 year

Used to immobilize microtubules via a biotin-neutravidin-biotin linkage; store in small aliquots

Kappa-casein (KC)

5 mg/mL

1X BRB80

-80°C

0.5 mg/mL

0.5 mg/mL

up to 2 years

Used to block the imaging chamber surface; Store in small aliquots; On day of experiment, set a small volume aside at room temperature

Bovine Serum Albumin (BSA)

50 mg/mL

1X BRB80

-20°C

1 mg/mL (in DB)

N/A

up to 2 years

store in small aliquots

Catalase

3.5 mg/mL

1X BRB80

-80°C

350 µg/mL (in OSF)

35 µg/mL

up to 2 years

component of oxygen scavenging mix; store in small aliquots

Glucose oxidase

40 mg/mL

1X BRB80

-80°C

4 mg/mL (in OSF)

0.4 mg/mL

up to 2 years

component of oxygen scavenging mix; store in small aliquots

Tubulin

Lyophilized

N/A

4°C

10 mg/mL

2.12 mg/mL (in tubulin mix)

up to 1 year

Once tubulin is in solution, keep it cold to avoid polymerization.

Adenosine Triphosphate (ATP)

100 mM

ultrapure water

-20°C

10 mM

1 mM

6 months

Prepare solution in filter-sterilized water, adjust pH to ~7.0, and freeze in small aliquots.

Guanosine Triphosphate (GTP)

100 mM

ultrapure water

-20°C

10 mM

1.29 mM (in tubulin mix)

6 months

Prepare solution in filter-sterilized water, adjust pH to ~7.0, and freeze in small aliquots.

Guanosine-5'-[(α,β)-methyleno] triphosphate (GMPCPP)

10 mM

ultrapure water

-20°C

10 µM

0.5 µM

6 months

Dithiothreitol (DTT)

1 M

sterile water

-20°C

1 mM

N/A

up to 2 years

Tris(2-carboxyethyl) phosphine (TCEP)

0.5 M

filter-sterilized water

Room temperature

5 mM

N/A

up to 2 years

Methylcellulose

1%

sterile water

Room temperature

0.8% (in MBMC)

0.21% (in tubulin mix)

up to 1 year

Dissolve methylcellulose by slowly adding it to near-boiling water. Allow to cool while stirring continuously.

Beta-mercaptoethanol (BME)

143 mM

sterile water

Room temperature

14.3 mM (in OSF)

1.43 mM

up to 5 years

143 mM is a 1:100 dilution of stock BME

Glucose

150 mg/mL

1X BRB80

-80°C

15 mg/mL (in OSF)

1.5 mg/mL

up to 2 years

Add to OSM immediately before use

(±)-6-Hydroxy-2,5,7,8-tetramethyl chromane-2-carboxylic acid (Trolox)

10mM

1X BRB80

-80°C

10 mM

1 mM

up to 1 year

Does not fully dissolve. Add some NaOH, stir for ~4 hours, and filter sterilize before use

mPEG-Succinimidyl Valerate, MW 5,000

powder

N/A

-20°C

333 mg/mL (in 0.1 M sodium bicarbonate)

324 mg/mL (in 0.1 M sodium bicarbonate)

6 months

Prepare ~34 mg aliquots, marking each tube with an exact weight of powder. Pass nitrogen gas over the solid, seal tubes with parafilm, and store at -20°C in a container with desiccant.

Biotin-PEG-SVA, MW 5,000

powder

N/A

-20°C

111 mg/mL (in 0.1 M sodium bicarbonate)

3.24 mg/mL (in 0.1 M sodium bicarbonate)

6 months

Prepare ~3 mg aliquots, marking each tube with an exact weight of powder. Pass nitrogen gas over the solid, seal tubes with parafilm, and store at -20°C in a container with desiccant.

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.

Reagent	Bright mix (µL)	Order of addition	Bright mix + biotin (µL)	Order of addition
Fluorescent tubulin, 10 mg/mL	2	6	2	7
Biotin-tubulin, 10 mg/mL	0	N/A	2	6
Unlabeled tubulin, 10 mg/mL	20	5	18	5
GMPCPP, 10 mM	30	4	30	4
DTT, 0.2 M	0.7	3	0.7	3
5X BRB80	26.4	2	26.4	2
sterile water	52.9	1	52.9	1
Total Volume (µL)	132		132

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.

Step	Reagent	Volume (µL)	Incubation time (minutes)
1	Neutravidin	7.5	5
2	MB-cold	10	-
3	κ-casein	7.5	2
4	MB-warm	10	-
5	Biotinylated microtubule (diluted in MB-warm)	10	10
6	MB-warm	10	-
7	Warm κ-casein	7.5	2
8	2 nM PRC1 diluted in κ-casein	10	5
9	Non-Biotinylated Microtubule	10	10
10	MB-warm x 2	10	-
11	Assay mix	10	-
Attached seeds are stable for around 20 minutes at this point

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

Reagent	Volume (µL)
Recycled tubulin, 10 mg/mL	10
MB-Cold	10.3
MBMC	13.7
BRB80-DTT	3.4
GTP, 10 mM	6.7
ATP, 10 mM (If using kinesins)	6.7
Fluorescently labeled tubulin, 10 mg/mL	1 (Resuspend lyophilized labeled tubulin in cold BRB80-DTT)

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.

Reagent	Volume (µL)
Soluble tubulin mix	4
OSF	1
Trolox (if using microtubules labeled with a readily-photobleaching fluorophore)	1
ATP, 10 mM (If using kinesins)	1
PRC1 (or crosslinker of choice)	1
Proteins of interest	X
MB-cold	2-X

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

Representative Results

The experiment described above was performed using 647 nm fluorophore-labeled biotinylated microtubules, 560 nm fluorophore-labeled non-biotinylated microtubules, and 560 nm fluorophore-labeled soluble tubulin mix. Microtubules were crosslinked by the crosslinker protein PRC1 (GFP-labeled). After surface-immobilized bundles and single microtubules were generated (step 5.11), the imaging chamber was mounted on a TIRF 100X 1.49 NA oil objective and viewed in the 560 nm and 647 nm fluorescence channels. Single microtubules were identified by their fluorescence signal in the 647 nm channel. Microtubules with fluorescence signals in both channels were identified as pre-formed bundles (Figure 4). If experiments are performed with biotinylated and non-biotinylated microtubules with the same fluorescent label, detected fluorescence intensities for bundles will be around two-fold or higher than that of single microtubules. Based on the proportion and density of each population, the concentration of microtubule seeds in steps 5.6 and 5.10 can be optimized.

Figure 4: Identification of single microtubules and crosslinked microtubules in the field of view. Representative field of view showing 647 nm (left), 560 nm (center), and merged (right) channels. Single microtubules (yellow arrowheads) and bundles (white arrowheads) are indicated in the merged channel. Scale bar represents 2 µm. Please click here to view a larger version of this figure.

Video 1: Dynamics of single microtubules and PRC1-crosslinked bundles. Representative video showing microtubule dynamics, with 647 nm fluorophore-labeled biotinylated microtubule seeds (blue), 560 nm fluorophore-labeled non-biotinylated microtubule seeds and 560 nm fluorophore-labeled soluble tubulin (red), and GFP-labeled PRC1 (green). Single and crosslinked microtubules are indicated by white and yellow arrows, respectively. The movie was recorded over 10 min (61 frames) and displayed at a rate of 12 frames/second. Assay conditions: 0.5 nM GFP-PRC1, 50 mM KCl and 37 °C. Scale bar: 5 µm. Please click here to download this Video.

After an imaging sequence has been acquired, analyze the video to ensure that microtubules are dynamic (Video 1). Adjust assay components (tubulin volume in soluble tubulin mix, nucleotide stocks, protein concentrations) according to observations. For example, if microtubules do not polymerize, increase concentration of soluble tubulin and/or GTP in soluble tubulin mix. Similarly, increase working concentrations of fluorescently labeled MAPs if they are not visible on microtubules, and decrease concentrations if their background fluorescence intensity in the field of view is comparable to their intensity on microtubules. When visualizing motile motor proteins, increase ATP concentrations if motors do not exhibit motility on microtubules. Adjust Laser intensity for the excitation channels corresponding to microtubule fluorescence to ensure that differences in fluorescence intensity between single microtubules and bundles can be captured within the dynamic range of the detector.

Video 2: Differences in dynamics of single microtubules and PRC1-crosslinked bundles in the presence of two MAPs: CLASP1 and Kif4A. Representative video showing microtubule dynamics, with 647 nm fluorophore-labeled biotinylated microtubule seeds (blue), and 560 nm fluorophore-labeled non-biotinylated microtubule seeds and 560 nm fluorophore-labeled soluble tubulin (red). Single and crosslinked microtubules are indicated by white and yellow arrows, respectively. The movie was recorded over 20 min (121 frames) and displayed at a rate of 20 frames/second. Assay conditions: 200 nM CLASP1-GFP, 0.5 nM PRC1, and 10 nM Kif4A. Scale bar: 2 µm. Video is reproduced from reference¹¹. Please click here to download this Video.

In the representative example shown in Video 2, a field of view containing single microtubules and crosslinked bundles is shown. It is found that under these assay conditions (assay mix containing 0.5 nM PRC1, 50 mM KCl, and MAPs of interest: 200 nM GFP-labeled CLASP1, 10 nM Kinesin Kif4A), single microtubules elongate over the course of the assay, whereas the growth of crosslinked microtubules is stalled.

For quantitative analysis of microtubule dynamics, open microscopy files in the FIJI software, and select single microtubules and bundles for analysis. Use the following criteria to exclude single microtubules and bundles from further analysis: exclude microtubules or bundles (i) found at the edges of the field of view, (ii) obscured by protein aggregates, or (iii) whose filaments move in the z-direction out of the TIRF range. Parameters of microtubule dynamics, such as length, growth rate, rescue frequency, and catastrophe frequency, can be obtained by constructing and analyzing kymographs for each single microtubule or microtubule pair^11,15.

Discussion

The experiment described here significantly expands the scope and complexity of conventional microtubule reconstitution assays, which are traditionally performed on single microtubules or on one type of array. The current assay provides a method to simultaneously quantify and compare the regulatory MAP activity on two populations, namely, single microtubules and crosslinked bundles. Further, this assay allows for the examination of two types of bundles: those that are pre-formed from stable seeds before the initiation of dynamics, and those that are newly formed when two growing ends encounter each other and get crosslinked (Figure 3). Moreover, in addition to conventional experimental variables, such as protein concentrations and buffer conditions, these assays enable the evaluation of the effects of geometrical features of microtubule arrays, such as the lengths and angles between adjacent filaments in a bundle, which are emerging as important determinants of microtubule dynamics and MAP activity¹⁶.

In order to extend this experimental method for the in vitro reconstitution of multiple microtubule-based structures, the following key issues need to be addressed: (i) PRC1 preferentially crosslinks microtubules that are oriented anti-parallel to each other. While such bundles are found at the cell center during mitosis, bundles of parallel microtubules are a common feature in other microtubule-based structures within neuronal axons and the mitotic spindle. The protocol described above can be readily adapted to generate crosslinked parallel microtubules using recombinant crosslinkers such as Kinesin-1 and TRIM46^10,17. (ii) In these reconstitution assays, differences in fluorescence intensity can be used to distinguish between single microtubules and pairs of microtubules^11,15. Under the experimental conditions used here, intensity analyses indicate that most bundles contain two crosslinked microtubules, and line scan analyses provide information on their relative positioning. However, when there are more than two or three filaments in a bundle, the spatial resolution of standard TIRF-based imaging systems hinders identification of the ends and polarity of individual microtubules (~25 nm diameter)¹⁸. Moreover, while it is possible to identify the plus-ends of crosslinked anti-parallel microtubules from the direction of their growth, distinguishing the ends of cross-linked parallel microtubules growing in the same direction is hindered by the spatial resolution of optical microscopy. An extension of the experiment described here is to use polarity-marked microtubules or microtubule tip-binding proteins to position individual microtubules. For bundles with tens of microtubules, complementing the high temporal resolution of TIRF-based assays with techniques that have high spatial resolution, such as Atomic Force Microscopy¹⁹, promises to yield new insights into the dynamics of individual microtubules within a bundle.

Materials

Name	Company	Catalog Number	Comments
(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)	Sigma Aldrich	238813
1,4-piperazinediethanesulfonic acid (PIPES)	Sigma Aldrich	P6757
18x18 mm #1.5 coverslips	Electron Microscopy Sciences	63787
2-Mercaptoethanol (BME)	Sigma Aldrich	M-6250
24x60 mm #1.5 coverslips	Electron Microscopy Sciences	63793
405/488/560/647 nm Laser Quad Band	Chroma	TRF89901-NK
Acetone	Sigma Aldrich	320110
Adenosine 5'-triphosphate disodium salt hydrate (ATP)	Sigma Aldrich	A7699-5G
Avidin, NeutrAvidin® Biotin-binding Protein (Molecular Probes®)	Thermo Fischer Scientific	A2666
Bath sonicator: Branson 2800 Cleaner	Branson	CPX2800H
Beckman Coulter Polycarbonate Thickwall Tubes, 11 x 34 mm	Beckman-Coulter	343778
Beckman Coulter Polycarbonate Thickwall Tubes, 8 x 34 mm	Beckman-Coulter	343776
Biotin-PEG-SVA, MW 5,000	Laysan Bio	#Biotin-PEG-SVA-5000
Bovine Serum Albumin (BSA)	Sigma Aldrich	2905
Catalase	Sigma Aldrich	C40
Corning LSE Mini Microcentrifuge, AC100-240V	Corning	6670
Delicate Task Wipes	Kimtech	34120
Dithiothreitol (DTT)	GoldBio	DTT10
Emission filter	Chroma	ET610/75m
Ethanol (200-proof)	Decon Labs	2705
Ethylene glycol tetraacetic acid (EGTA)	Sigma Aldrich	3777
Glucose Oxidase	Sigma Aldrich	G2133
GMPCPP	Jena Bioscience	NU-405
Guanosine 5'-triphosphate sodium salt hydrate (GTP)	Sigma Aldrich	G8877
Hellmanex III detergent	Sigma Aldrich	Z805939
Immersion oil, Type A	Fisher Scientific	77010
Kappa-casein	Sigma Aldrich	C0406
Lanolin	Fisher Scientific	S25376
Lens Cleaning Tissue	ThorLabs	MC-5
Magnesium Chloride (MgCl2)	Sigma Aldrich	M9272
Methylcellulose	Sigma Aldrich	M0512
Microfuge 16 Benchtop Centrifuge	Beckman-Coulter	A46474
Microscope Slides, Diamond White Glass, 25 x 75mm, 90° Ground Edges, WHITE Frosted	Globe Scientific	1380-50W
mPEG-Succinimidyl Valerate, MW 5,000	Laysan Bio	#NH2-PEG-VA-5K
Optima™ Max-XP Tabletop Ultracentrifuge	Beckman-Coulter	393315
Paraffin	Fisher Scientific	P31-500
PELCO Reverse (self-closing), Fine Tweezers	Ted Pella	5377-NM
Petrolatum, White	Fisher Scientific	18-605-050
Plasma Cleaner, 115V	Harrick Plasma	PDC-001
Potassium Hydroxide (KOH)	Sigma Aldrich	221473
Sodium bicarbonate	Sigma Aldrich	S6014
Sucrose	Sigma Aldrich	S7903
Thermal-Lok 1-Position Dry Heat Bath	USA Scientific	2510-1101
Thermal-Lok Block for 1.5 and 2.0 mL Tubes	USA Scientific	2520-0000
Thermo Scientific™ Pierce™ Bond-Breaker™ TCEP Solution, Neutral pH; 500mM	Thermo Fischer Scientific	PI-77720
TIRF 100X NA 1.49 Oil Objective	Nikon	CFI Apochromat TIRF 100XC Oil
TIRF microscope	Nikon	Eclipse Ti
TLA 120.1 rotor	Beckman-Coulter	362224
TLA 120.2 rotor	Beckman-Coulter	357656
Tubulin protein (>99% pure): porcine brain	Cytoskeleton	T240
Tubulin Protein (Biotin): Porcine Brain	Cytoskeleton	T333P
Tubulin protein (fluorescent HiLyte 647): porcine brain	Cytoskeleton	TL670M
Tubulin protein (X-rhodamine): bovine brain	Cytoskeleton	TL620M
VECTABOND® Reagent, Tissue Section Adhesion	Vector Biolabs	SP-1800-7
VWR® Personal-Sized Incubator, 120V, 50/60Hz, 0.6A	VWR	97025-630