Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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 potassium salts and NOT sodium salts³⁰.

1. Prepare 1 L of complete medium: Dulbecco’s modified Eagle medium (DMEM), w....

Results

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 motors^25<.......

Discussion

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 years^16,17. 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.......

Materials

List of materials used in this article

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