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This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions.
One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The “tubulin code” is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs.
To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps.
We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.
Microtubules play critical roles in many cellular processes. They give cells their shape, build meiotic and mitotic spindles for chromosome segregation, and serve as tracks for intracellular transport. To perform these diverse functions, microtubules organize themselves in different ways. One of the intriguing questions in the field is to understand the molecular mechanisms that allow the structurally and evolutionarily conserved microtubules to adapt to this plethora of organizations and functions. One potential mechanism is the diversification of microtubules, which is defined by the concept known as the ‘tubulin code’1,
Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines.
1. Preparation of Reagents for Tubulin Purification
NOTE: All the buffers used for tubulin purification should contain potassiu.......
The main goal of this method is to produce high-quality, assembly-competent tubulin in quantities sufficient to perform repeated in vitro experiments with the purified components. Microtubules assembled from this tubulin can be used in reconstitution assays based on the total internal reflection fluorescence (TIRF) microscopy technique with either dynamic or stable microtubules, in experiments testing microtubule dynamics, interactions with MAPs or molecular motors, and force generation by the motors25<.......
The method described here provides a platform to rapidly generate high-quality, assembly-competent tubulin in medium-large quantities from cell lines and single mouse brains. It is based on the gold-standard protocol of tubulin purification from bovine brains used in the field for many years16,17. One particular advantage of the approach is the use of suspension cultures of HeLa S3 cells, which, once established, yields large amounts of cells while requiring litt.......
This work was supported by the ANR-10-IDEX-0001-02, the LabEx Cell’n’Scale ANR-11-LBX-0038 and the Institut de convergence Q-life ANR-17-CONV-0005. CJ is supported by the Institut Curie, the French National Research Agency (ANR) awards ANR-12-BSV2-0007 and ANR-17-CE13-0021, the Institut National du Cancer (INCA) grant 2014-PL BIO-11-ICR-1, and the Fondation pour la Recherche Medicale (FRM) grant DEQ20170336756. MMM is supported by the Fondation Vaincre Alzheimer grant FR-16055p, and by the France Alzheimer grant AAP SM 2019 n°2023. JAS was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodow....
Name | Company | Catalog Number | Comments |
1 M MgCl2 | Sigma | #M1028 | |
1-L cell culture vessels | Techne F7610 | Used for spinner cultures. Never stir the empty spinner bottles. When spinner bottles are in the cell culture incubator, always keep the lateral valves of spinner bottles slightly open to facilitate the equilibration of media with incubator’s atmosphere. After use, fill the spinner bottles immediately with tap water to avoid drying of remaining cells on the bottle walls. Wash the bottles with deionised water, add app 200 ml of deionised water and autoclave. Under a sterile cell culture hood remove the water and allow the bottles to dry completely, still under the hood, for several hours. Never use detergents for cleaning the spinner bottles because any trace amounts of the detergent can be deleterious to the cells. | |
1.5- and 2-ml tubes | |||
14-ml round-bottom tubes | |||
15-cm-diameter sterile culture dishes | |||
15-ml screw-cap tubes | |||
2-mercaptoethanol | Sigma | #M3148 | 2-mercaptoethanol is toxic and should be used under the hood. |
4-(2-aminoethyl)-benzenesulfonyl fluoride | Sigma | #A8456 | |
40% Acrylamide | Bio-Rad | #161-0140 | |
5-, 10- 20-ml syringes | |||
5-ml, 10-ml, 25-ml sterile pipettes | |||
50-ml screw-cap tubes | |||
Ammonium persulfate (APS) | Sigma | #A3678 | |
Anti-alpha-tubulin antibody, 12G10 | Developed by J. Frankel and M. Nelson, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by the University of Iowa | dilution: 1/500 | |
Anti-glutamylated tubulin antibody, GT335 | AdipoGen | #AG-20B-0020 | dilution: 1/20,000 |
Aprotinin | Sigma | #A1153 | |
Balance (0.1 – 10 g) | |||
Beckman 1-l polypropylene bottles | For collecting spinner cultures | ||
Beckman Avanti J-26 XP centrifuge | For collecting spinner cultures | ||
Biological stirrer | Techne MCS-104L | Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells | |
Bis N,N’-Methylene-Bis-Acrylamide | Bio-Rad | #161-0201 | |
Blender IKA Ultra-Turrax® | For lysing brain tissue, use 5-mm probe, with the machine set at power 6 or 7. Blend the brain tissue 2-3 times for 15 s on ice. | ||
Bovine serum albumin (BSA) | Sigma | #A7906 | |
Bromophenol blue | Sigma | #1.08122 | |
Carboxypeptidase A (CPA) | Sigma | #C9268 | Concentration: 1.7 U/µl |
Cell culture hood | |||
Cell culture incubator set at 37°C, 5% CO2 | |||
Dimethyl sulfoxide (DMSO) | Sigma | #D8418 | DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection. |
DMEM medium | Life Technologies | #41965062 | |
DTT, DL-Dithiothreitol | Sigma | #D9779 | |
EDTA | Euromedex | #EU0007-C | |
EGTA | Sigma | #E3889 | |
Ethanol absolute | Fisher Chemical | #E/0650DF/15 | |
Fetal bovine serum (FBS) | Sigma | #F7524 | |
French pressure cell press | Thermo electron corporation | #FA-078A | with a #FA-032 cell; for lysing big amounts of cells. Set at medium ratio, and the gauge pressure of 1,000 psi (corresponds to 3,000 psi inside the disruption chamber). |
Glycerol | VWR Chemicals | #24388.295 | |
Glycine | Sigma | #G8898 | |
GTP | Sigma | #G8877 | |
Heating block | Stuart | #SBH130D | |
Hela cells | ATCC® CCL-2™ | ||
Hela S3 cells | ATCC | ATCC® CCL-2.2™ | |
Hydrochloric acid (HCl ) | VWR | #20252.290 | |
Inverted microscope | With fluorescence if cell transfection is to be verified | ||
Isopropanol | VWR | #20842.298 | |
jetPEI | Polyplus | #101 | |
JLA-8.1000 rotor | For collecting spinner cultures | ||
KOH | Sigma | #P1767 | KOH is corrosive and causes burns; use eye and skin protection. |
L-Glutamine | Life Technologies | #25030123 | |
Laboratory centrifuge for 50-ml tubes | Sigma | 4-16 K | |
Leupeptin | Sigma | #L2884 | |
Liquid nitrogen | |||
Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips | |||
Micropestles | Eppendorf | #0030 120.973 | |
Mouse brain tissue | Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines. | ||
Needles 18G X 1 ½” (1.2 X 38 mm | Terumo | #18G | |
Needles 20G X 1 ½” (0.9 X 38 mm | Terumo | #20G | |
Needles 21G X 4 ¾” (0.8 X 120 mm | B.Braun | #466 5643 | |
Parafilm | |||
PBS | Life Technologies | #14190169 | |
Penicillin-Streptomycin | Life Technologies | #15140130 | |
pH-meter | |||
Phenylmethanesulfonyl fluoride (PMSF) | Sigma | #P7626 | PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions. |
PIPES | Sigma | #P6757 | |
Pipette-boy | |||
Rotors | Beckman 70.1 Ti; TLA-100.3; and TLA 55 | ||
SDS-PAGE electrophoresis equipment | Bio-Rad | #1658001FC | |
SDS, Sodium dodecyl sulphate | VWR | #442444H | For preparing Laemmeli buffer |
SDS, Sodium dodecyl sulphate | Sigma | #L5750 | For preparing 'TUB' SDS-PAGE gels |
Sonicator | Branson | #101-148-070 | Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at “Output control” 1, “Duty cycle” 10% and time depending on the cell type used. |
Tabletop centrifuge for 1.5 ml tubes | Eppendorf | 5417R | |
TEMED, N, N, N′, N′-Tetramethylethylenediamine | Sigma | #9281 | |
Trichostatin A (TSA) | Sigma | #T8552 | |
Triton X-100 | Sigma | #T9284 | |
Trizma base (Tris) | Sigma | #T1503 | |
Trypsin | Life Technologies | #15090046 | |
Ultracentrifuge rotors | TLA-55, TLA-100.3 and 70.1 Ti rotors | Set at 4°C or 30°C based on the need of the experiment | |
Ultracentrifuge tubes | Beckman | #357448 | for using with TLA-55 rotor |
Ultracentrifuge tubes | Beckman | #349622 | for using with TLA-100.3 rotor |
Ultracentrifuge tubes | Beckman | #355631 | for using with 70.1 Ti rotor |
Ultracentrifuges | Beckman | Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent) | Set at 4°C or 30°C based on the need of the experiment |
Vortex mixer | |||
Water bath equipped with floaters or tube holders | Set at 30°C |
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