Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

Summary

Microtubules, which are tubulin polymers, play a crucial role as a cytoskeleton component in eukaryotic cells and are known for their dynamic instability. This study developed a method for fractionating microtubules to separate them into stable microtubules, labile microtubules, and free tubulin to evaluate the stability of microtubules in various mouse tissues.

Abstract

Microtubules, composed of α/β-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects.

Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues.

The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 × g) to remove debris. The first ultracentrifugation step (100,000 × g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 × g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Introduction

Microtubules (MTs) are elongated tubular structures comprising protofilaments consisting of α/β-tubulin heterodimer subunits. They play essential roles in various cellular processes such as cell division, motility, shape maintenance, and intracellular transport, making them integral components of the eukaryotic cytoskeleton¹. The minus-end of MTs, where the α-tubulin subunit is exposed, is relatively stable, whereas the plus-end, where the β-tubulin subunit is exposed, undergoes dynamic depolymerization and polymerization². This continuous cycle of tubulin dimer addition and dissociation at the plus-end, referred to as dynamic instability, results in a repetitive process of rescue and catastrophe³. MTs exhibit focal domains with localized variations in dynamic instability, including stable and labile domains⁴.

Precise control of the dynamic instability of MTs is crucial for numerous cellular functions, particularly in neurons characterized by intricate morphologies. The adaptability and durability of MTs play a vital role in the development and proper functioning of nerve cells^5,6,7. The dynamic instability of MTs has been found to be associated with various post-translational modifications (PTMs) of tubulin, such as acetylation, phosphorylation, palmitoylation, detyrosination, delta 2, polyglutamine oxidation, and polyglycylation. Additionally, the binding of microtubule-associated proteins (MAPs) serves as a regulatory mechanism⁸. PTMs, excluding acetylation, predominantly occur in the tubulin carboxy-terminal region situated on the external surface of MTs. These modifications create diverse surface conditions on MTs, influencing their interaction with MAPs and ultimately governing MT stability⁹. The presence of a carboxy-terminal tyrosine residue in α-tubulin is indicative of dynamic MTs, which are rapidly replaced by the free tubulin pool. Conversely, detyrosination of the carboxy terminus and acetylation of Lys40 signify stable MTs with reduced dynamic instability^9,10.

The PTMs of tubulin have been extensively employed in experiments to assess the dynamics and stability of MTs^{5,7,11,12,13,14,15}. For instance, in cell culture studies, tubulins can be segregated into two pools: the free tubulin pool and the MT pool. This is achieved by releasing free tubulin through cell permeabilization before fixing the remaining MTs^{15,16,17,18,19}. Biochemical methods involve the use of chemical MT stabilizers that safeguard MTs from catastrophe, enabling the separation of MTs and free tubulin through centrifugation^20,21,22. However, these procedures do not differentiate between stable and less stable (labile) MTs, thereby rendering it impossible to quantify MTs or soluble tubulin in tissues like the brain. Consequently, evaluating MT stability in organisms under physiological and pathological conditions has proven to be challenging. To address this experimental limitation, we have developed a novel technique for precisely separating MTs and free tubulin in mouse tissue²³.

This unique MT fractionation method involves tissue homogenization under conditions that maintain tubulin status in tissues and two-step centrifugation to separate stable MTs, labile MTs, and free tubulin. This simple procedure can be applied to broad studies, including basic research on MTs and MAPs in living organisms, physiological and pathological analyses of health and diseases associated with MT stability, and developing drugs and other therapeutics that target MTs.

Protocol

1. MT fractionation method

NOTE: All experiments performed in this study were approved by the Animal Ethics Committee of Doshisha University. C57BL/6J mice of either sex, 3-4 months of age, were used here. In this protocol, dissected tissues, e.g., brain, liver, or thymus, were immediately homogenized in ice-cold microtubule stabilizing buffer (MSB), which contained Taxol (MT stabilizer) at a concentration that prevented not only depolymerization but also repolymerization of MT. The homogenate was separated into three fractions by a two-step ultracentrifugation process (Figure 1). All steps in this protocol were completed without interruption in a cool-temperature environment, and the tissues and fractions were not frozen until they were dissolved in sodium dodecyl sulfate (SDS)-sample buffer.

1. Preparation of MSB and microtubes
   1. To prepare MSB, mix the following reagents: 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.8 (neutralized by KOH), 10% glycerol, 0.1 mM DTT, 1 mM MgSO₄, 1 mM EGTA, 0.5% Triton X-100, phosphatase inhibitors (1 mM NaF, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 0.5 µM okadaic acid), 1x protease inhibitor cocktail, and protease inhibitors (0.1 mM PMSF, 0.1 mM DIFP, 1 µg/mL pepstatin, 1 µg/mL antipain, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 50 µg/mL TLCK) (see Table of Materials) and denote as MSB(-).
   2. Just before tissue dissection, add 10 µM Taxol and 2 mM GTP (see Table of Materials) to MSB(-). This buffer is denoted as MSB(+). Prepare the buffer on the day of use and keep it on ice.
   3. Prepare the microtubes for sampling. Empty 2.0 mL microtube for homogenate storage; empty 1.5 mL microtube for supernatant1 (S1) lysate, precipitate2 (P2) sample, and precipitate3 (P3) sample storage; 1.5 mL microtube with 1 mL of ice-cold phosphate-buffered saline (PBS) for dissected tissue storage; 1.5 mL microtube with 200 µL of 2x SDS-sample buffer (0.16 M Tris pH 6.8; 20% glycerol; 2% 2-mercaptoethanol; 4% SDS) for S1 sample and supernatant3 (S3) sample storage; and centrifugation microtube for TLA55 and TLA120.2 centrifuge rotors (see Table of Materials). Label all tubes and place them on ice.
2. Mouse tissue homogenization
   1. Prepare a chilled table for tissue dissection. First, fill a box with crushed ice and place two Petri dishes on ice, one with the inner side up and the other with the outer side. Fill ice-cold PBS in one dish for transient wash and storage of dissected tissues. Lay filter paper moistened with PBS on another dish turned over.
   2. To sacrifice a mouse, perform cervical dislocation under deep anesthesia with a mixed anesthetic of butorphanol, midazolam, and medetomidine. Then, immediately dissect the tissues, e.g., brain, liver, or thymus, and wash them with ice-cold PBS in a Petri dish.
      NOTE: Any type of soft tissue can be analyzed by this method. However, the size of the tissue is limited by the recommended volume range of the homogenizer used. For example, if a 2 mL volume of homogenizer is used, 50-100 mg of tissue is recommended.
   3. After weighing the 1.5 mL microtubes filled with PBS for dissected tissue storage, cut out and store tissues inside the microtubes, and reweigh each microtube. Each tissue's wet weight can be calculated by subtracting the weight of the tube before and after the tissue is added.
   4. Immediately homogenize the tissue in ice-cold MSB(+) with a chilled homogenizer (see Table of Materials). The volume of MSB(+) was 19 times (µL) the tissue wet weight (mg). Perform homogenization with 20 strokes until the tissue pieces disappear.
      NOTE: For example, 1,900 µL of MSB(+) is used for 100 mg of tissue. Since the volume of MSB(+) to be added must be adjusted for each wet weight of the tissue pieces analyzed, it is necessary to weigh each tissue piece accurately.
3. Centrifugation of the mouse tissue homogenates
   1. Move the whole homogenate to a 2 mL microtube with a Pasteur pipette and centrifuge at 2,400 × g for 3 min at 2 °C to remove the debris via precipitation.
   2. Transfer the whole supernatant (S1 fraction) to a new 1.5 mL microtube and vortex. Then, aliquot 200 µL of S1 fraction into a centrifugation microtube and centrifuge at 100,000 × g using a TLA-55 rotor for 20 min at 2 °C to obtain the relatively large molecular weight proteins as a precipitate (P2 fraction).
      NOTE: The volume of the sample subjected to the ultracentrifugation steps affects the radius of centrifugation and the precipitation efficiency of the molecules. Keep the sample volume at 200 µL or less after this step to prevent inaccurate fractionation.
   3. Further centrifuge all resultant supernatant (S2 fraction) at 500,000 × g using a TLA-120.2 rotor for 60 min at 2 °C to separate the insoluble protein complexes in the precipitate (P3 fraction) from soluble proteins in the supernatant (S3 fraction).
   4. Add 400 µL of 1x SDS-sample buffer (0.08 M Tris pH 6.8; 10% glycerol; 1% 2-mercaptoethanol; 2% SDS) to the P2 and P3 fraction tubes and briefly sonicated to dissolve the precipitate. Transfer these fraction samples to an empty 1.5 mL microtube.
   5. Dissolve the total S3 fraction in 200 µL of 2x SDS-sample buffer.
   6. Mix the remaining S1 fractions with an equal volume of 2x SDS sample buffer for use as a standard curve for Western blotting.
   7. Boil all these samples at 100 °C for 3 min. After the samples have cooled to room temperature, store the samples at -20 °C.
4. Quantification of proteins in each fraction
   1. Quantify proteins in the P2, P3, and S3 fractions by Western blotting. First, use 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins from properly diluted the P2, P3, and S3 fractions, and serially diluted S1 sample from any individual as a standard curve. Then, electroblot the samples onto polyvinylidene fluoride membranes (see Table of Materials).
      ​NOTE: The dilution ratio of each fraction depends on the concentration of an objective protein and the reactivity of the antibody. (e.g., α-tubulin, TUBB3, β-tub, and tyrosinated tubulin in brain tissue: S3 = 1/400, P3 = 1/2,000, P2 = 1/2,000, S1 = 1/50,000, 1/20,000, 1/10,000, 1/5,000, 1/2,000; acetylated tubulin in brain tissue: S3 = 1/200, P3 = 1/400, P2 = 1/8,000, S1 = 1/100,000, 1/40,000, 1/20,000, 1/10,000, 1/4,000; α-tubulin in liver: S3 = 1/20, P3 = 1/100, P2 = 1/20, S1 = 1/50,000, 1/20,000, 1/10,000, 1/5,000, 1/2,000; α-tubulin in thymus: S3 = 1/100, P3 = 1/400, P2 = 1/20, S1 = 1/50,000, 1/20,000, 1/10,000, 1/5,000, 1/2,000 dilution of tissue concentration).
   2. Block the membrane with 5% skimmed milk in Tris-buffered saline (50 mM Tris-HCl pH 7.6; 152 mM NaCl) with 0.1% Tween 20 (TBS-T) for over 30 min.
   3. Immerse the membrane in TBS-T containing primary antibody (see Table of Materials) for over 2 h. After that, wash the membrane with TBS-T for 3 min (3 times).
   4. Label primary antibody using HRP-conjugated secondary antibodies (see Table of Materials) in TBS-T for over 1 h. After that, wash the membrane with TBS-T for 3 min (3 times).
   5. Develop the membranes with Enhanced Chemiluminescence reagent. Then, analyze the bands of interest with a luminescent image analyzer (see Table of Materials).
   6. Quantify the protein band intensities using image analysis software (see Table of Materials) and create a standard curve by plotting the dilution units of the diluted S1 samples used for the standard curve along the X-axis and the band intensities along the Y-axis.
   7. Read the protein concentration (unit) corresponding to the diluted fraction samples. Multiply the concentration read with the sample dilution factor to obtain a protein unit in each fraction. Divide the measured unit of each fraction by the total protein unit (P2 + P3 + S3) to obtain a percentage.

2. Evaluation of the properties of tubulin in each fraction

NOTE: This biochemical method provides three groups of tubulin complexes defined by sedimentation properties. Here, the status of tubulin complexes obtained in these fractions was identified based on the size of the complex and tubulin-PTMs. Complete all steps in this protocol without interruption in a cool-temperature environment but do not freeze the fraction samples until they are dissolved in the SDS-sample buffer.

1. Filter trap assay
   1. Filter the S2 and S3 fractions (500 µL each) using a 300 kDa ultrafiltration spin column (see Table of Materials). Perform 14,000 × g centrifugation at 2 °C until the whole supernatant is filtered, eluted proteins are collected into receiver tubes, and trapped proteins remain on the filter of reservoir tubes.
   2. Translate the whole filtrates (approximately 500 µL) in receiver tubes into new 1.5 mL microtubes and dissolve them in 500 µL of 2x SDS-sample buffer.
   3. Solubilize the residues on the filter of reservoir tubes in 1,000 µL of 1x SDS-sample buffer by pipetting and transferring the samples to a new 1.5 mL microtube.
   4. Boil the samples at 100 °C for 3 min. After the samples have cooled to room temperature, store the samples at -20 °C.
   5. Analyze the amounts of tubulin by Western blotting with DM1A (anti-α-tubulin antibody, see Table of Materials).
2. Size-exclusion chromatography
   1. Prepare the carrier buffer (0.1 M MES, pH 6.8; 10% glycerol; 1 mM MgSO₄; 1 mM EGTA; 0.1 mM DTT) supplemented with 1/10 concentration of protease and phosphatase inhibitors as described in step 1.1.1. Then, filter the solution and store it in a cold environment.
   2. Prepare a gel filtration chromatography column equipped with a preparative liquid chromatography system in a chromatography chamber (see Table of Materials) at 4 °C.
   3. Before injecting samples onto the column, flow 180 mL of the carrier buffer to wash the column. The flow rate is 1 mL/min for 3 h.
   4. Inject commercially available purified porcine tubulin (see Table of Materials) as a control or the S3 fraction from mouse brains (500 µL each) onto the column.
   5. Elute at a flow rate of 1.0 mL/min with carrier buffer. Collect the 1.5 mL fractions for 120 min. Monitor eluted proteins by absorbance at 280 nm. Keep the maximum pressure under 0.3 MPa.
   6. After vortexing the collected fractions, mix 50 µL of them with 50 µL of 2x SDS-sample buffer in a 1.5 mL microtube. Boil all samples at 100 °C for 3 min. After the samples have cooled to room temperature, store the samples at -20 °C.
   7. Analyze the amounts of tubulin by Western blotting with DM1A, anti-α-tubulin antibody and KMX-1, anti-β-tubulin antibody (see Table of Materials).

Results

Quantification of tubulin in the P2, P3, and S3 fractions from mouse brain by the MT fractionation method
Tubulin in mouse tissue was separated into the P2, P3, and S3 fractions by the MT fractionation method and quantified by Western blotting (Figure 1A). The precipitate of MTs that remained in the P2 fraction by ultracentrifugation at 100,000 × g for 20 min accounted for 34.86% ± 1.68% of total tubulin in a mouse brain. The supernatant (S2) was further centrif...

Discussion

The most significant task when investigating the status of tubulin in tissue from living organisms is preventing accidental MT polymerization or depolymerization during preparation. The stability of MTs in samples is affected by factors such as the concentration of Taxol in MSB, the proportion of tissue amount to buffer, and temperature during the process from tissue removal to homogenization and centrifugation. Therefore, the conditions were optimized in each step of the protocol for analyzing mouse tissue with a 20-fol...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 ML TUBE CASE OF 500	Beckman Coulter	357448
1A2	Sigma-Aldrich	T9028	1:5,000 dilution
2-(N-morpholino)ethanesulfonic acid (MES)	Nacalai Tesque	02442-44
300 kDa ultrafiltration spin column	Aproscience	PT-1013
6-11B1	Sigma-Aldrich	T7451	1:5,000 dilution
ÄKTAprime plus	Cytiva	11001313
anti-mouse IgG	Jackson ImmunoResearch	115-035-146	1:5,000 dilution
antipain	Peptide Institute Inc.	4062
aprotinin	Nacalai Tesque	03346-84
Chemi-Lumi One L	Nacalai Tesque	07880-54
Corning bottle-top vacuum filter system	Corning	430758	0.22µm 33.2cm² Nitrocellulose membrane
DIFP	Sigma-Aldrich	55-91-4
DIGITAL HOMOGENIZER HK-1	AS ONE	1-2050-11
DM1A	Sigma-Aldrich	T9026	1:5,000 dilution
DTT	Nacalai Tesque	14128-46
EGTA	Nacalai Tesque	37346-05
FluoroTrans W 3.3 Meter Roll	Pall Corporation	BSP0161
glycerol	Nacalai Tesque	17018-25
GTP	Nacalai Tesque	17450-61
HIGH SPEED REFRIGERATIOED MICRO CENTRIFUGE Kitman	TOMY	KITMAN-24
HiLoad 16/600 Superdex 200 pg column	Cytiva	28-9893-35
Image Gauge Software	FUJIFILUM Wako Pure Chemical Corporation
ImmunoStar LD	FUJIFILUM Wako Pure Chemical Corporation	292-69903
KMX-1	Millipore	MAB3408	1:5,000 dilution
LAS-4000 luminescent image analyzer	FUJIFILUM Wako Pure Chemical Corporation
leupeptin	Peptide Institute Inc.	43449-62
MgSO4	Nacalai Tesque	21003-75
Na3VO4	Nacalai Tesque	32013-92
NaF	Nacalai Tesque	31420-82
okadaic acid	LC Laboratories	O-2220
OPTIMA MAX-XP	Beckman Coulter	393315
pepstatin	Nacalai Tesque	26436-52
PMSF	Nacalai Tesque	27327-81
Polycarbonate Centrifuge Tubes for TLA120.2	Beckman Coulter	343778
Protease inhibitor cocktail (cOmplete™, EDTA-free)	Roche	11873580001
Purified tubulin	Cytoskeleton	T240
QSONICA Q55	QSonica	Q55
Taxol	LC Laboratories	P-9600
TLA-120.2 rotor	Beckman Coulter	357656
TLA-55 rotor	Beckman Coulter	366725
TLCK	Nacalai Tesque	34219-94
Triton X-100	Nacalai Tesque	12967-45
β-glycerophosphate	Sigma-Aldrich	G9422