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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.
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.
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 function2,3,4,5. 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 conditions8,9,10. 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.
1. Prepare reagents
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.
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
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
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.
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
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.
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 ...
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...
The authors declare no competing interests.
This work was supported by a grant from the NIH (no. 1DP2GM126894-01), and by funds from the Pew Charitable Trusts and the Smith Family Foundation to R.S. The authors thank Dr. Shuo Jiang for his contribution toward development and optimization of the protocols.
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 |
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