JoVE Logo
Authors
Contact Us

Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 cells5,6,7. 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 instability9,10.

The PTMs of tubulin have been extensively employed in experiments to assess the dynamics and stability of MTs5,7,11,12,13,14,15. 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 MTs15,16,17,18,19. Biochemical methods involve the use of chemical MT stabilizers that safeguard MTs from catastrophe, enabling the separation of MTs and free tubulin through centrifugation20,21,22. 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 fur...

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...

Disclosures

The authors have no conflicts of interest to report.

Acknowledgements

This work was supported in part by JST the establishment of university fellowships toward the creation of science technology innovation (A.HT.; JPMJFS2145), JST SPRING (A.HT.; JPMJSP2129), Grant-in-Aid for JSPS Fellows (A.HT.; 23KJ2078), a Grant-in-Aid for Scientific Research(B) JSPS KAKENHI (22H02946 for TM), a Grant-in-Aid for Scientific Research on Innovative Areas titled "Brain Protein Aging and Dementia Control" from MEXT (TM; 26117004), and by Uehara Research Fellowship from the Uehara Memorial Foundation (TM; 202020027). The authors declare no competing financial interests.

Materials

NameCompanyCatalog NumberComments
1.5 ML TUBE CASE OF 500Beckman Coulter357448
1A2Sigma-AldrichT90281:5,000 dilution
2-(N-morpholino)ethanesulfonic acid (MES)Nacalai Tesque02442-44
300 kDa ultrafiltration spin columnAprosciencePT-1013
6-11B1Sigma-AldrichT74511:5,000 dilution
ÄKTAprime plusCytiva11001313
anti-mouse IgGJackson ImmunoResearch115-035-1461:5,000 dilution
antipainPeptide Institute Inc.4062
aprotininNacalai Tesque03346-84
Chemi-Lumi One LNacalai Tesque07880-54
Corning bottle-top vacuum filter systemCorning4307580.22µm 33.2cm² Nitrocellulose membrane
DIFPSigma-Aldrich55-91-4 
DIGITAL HOMOGENIZER HK-1AS ONE1-2050-11
DM1ASigma-AldrichT90261:5,000 dilution
DTTNacalai Tesque14128-46
EGTANacalai Tesque37346-05
FluoroTrans W 3.3 Meter RollPall CorporationBSP0161
glycerolNacalai Tesque17018-25
GTPNacalai Tesque17450-61
HIGH SPEED REFRIGERATIOED MICRO CENTRIFUGE KitmanTOMYKITMAN-24
HiLoad 16/600 Superdex 200 pg columnCytiva28-9893-35
Image Gauge Software FUJIFILUM Wako Pure Chemical Corporation
ImmunoStar LD FUJIFILUM Wako Pure Chemical Corporation292-69903
KMX-1MilliporeMAB34081:5,000 dilution
LAS-4000 luminescent image analyzerFUJIFILUM Wako Pure Chemical Corporation
leupeptinPeptide Institute Inc.43449-62
MgSO4Nacalai Tesque21003-75
Na3VO4Nacalai Tesque32013-92
NaFNacalai Tesque31420-82
okadaic acidLC LaboratoriesO-2220 
OPTIMA MAX-XPBeckman Coulter393315
pepstatinNacalai Tesque26436-52
PMSFNacalai Tesque27327-81
Polycarbonate Centrifuge Tubes for TLA120.2Beckman Coulter343778
Protease inhibitor cocktail (cOmplete™, EDTA-free)Roche11873580001
Purified tubulin CytoskeletonT240
QSONICA Q55QSonicaQ55
TaxolLC LaboratoriesP-9600
TLA-120.2 rotorBeckman Coulter357656
TLA-55 rotorBeckman Coulter366725
TLCKNacalai Tesque34219-94
Triton X-100Nacalai Tesque12967-45
β-glycerophosphateSigma-AldrichG9422

References

  1. Janke, C., Magiera, M. M. The tubulin code and its role in controlling microtubule properties and functions. Nature Reviews Molecular Cell Biology. 21 (6), 307-326 (2020).
  2. Conde, C., Caceres, A. Microtubule assembly, organization and dynamics in axons and dendrites. Nature Reviews Neuroscience. 10 (5), 319-332 (2009).
  3. Mitchison, T., Kirschner, M. Dynamic instability of microtubule growth. Nature. 312 (5991), 237-242 (1984).
  4. Baas, P. W., Rao, A. N., Matamoros, A. J., Leo, L. Stability properties of neuronal microtubules. Cytoskeleton (Hoboken). 73 (9), 442-460 (2016).
  5. Challacombe, J. F., Snow, D. M., Letourneau, P. C. Dynamic microtubule ends are required for growth cone turning to avoid an inhibitory guidance cue. Journal of Neuroscience. 17 (9), 3085-3095 (1997).
  6. Kapitein, L. C., Hoogenraad, C. C. Building the neuronal microtubule cytoskeleton. Neuron. 87 (3), 492-506 (2015).
  7. Leo, L., et al. Vertebrate fidgetin restrains axonal growth by severing labile domains of microtubules. Cell Reports. 12 (11), 1723-1730 (2015).
  8. Janke, C. The tubulin code: molecular components, readout mechanisms, and functions. Journal of Cell Biology. 206 (4), 461-472 (2014).
  9. Wloga, D., Joachimiak, E., Fabczak, H. Tubulin post-translational modifications and microtubule dynamics. International Journal of Molecular Sciences. 18 (10), 2207 (2017).
  10. Baas, P. W., Black, M. M. Individual microtubules in the axon consist of domains that differ in both composition and stability. Journal of Cell Biology. 111 (2), 495-509 (1990).
  11. Cartelli, D., et al. Microtubule alterations occur early in experimental parkinsonism and the microtubule stabilizer epothilone D is neuroprotective. Scientific Reports. 3, 1837 (2013).
  12. Zhang, B., et al. Microtubule-binding drugs offset tau sequestration by stabilizing microtubules and reversing fast axonal transport deficits in a tauopathy model. Proceedings of the National Academy of Sciences of the United States of America. 102 (1), 227-231 (2005).
  13. Zhang, F., et al. Post-translational modifications of alpha-tubulin in Alzheimer disease. Translational Neurodegeneration. 4, 9 (2015).
  14. Miyasaka, T., et al. Curcumin improves tau-induced neuronal dysfunction of nematodes. Neurobiology of Aging. 39, 69-81 (2016).
  15. Fujiwara, H., et al. Inhibition of microtubule assembly competent tubulin synthesis leads to accumulation of phosphorylated tau in neuronal cell bodies. Biochemical and Biophysical Research Communications. 521 (3), 779-785 (2020).
  16. Vielkind, U., Swierenga, S. H. A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell. Histochemistry. 91 (1), 81-88 (1989).
  17. Kanai, Y., et al. Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA. Journal of Cell Biology. 109 (3), 1173-1184 (1989).
  18. Brown, A., Li, Y., Slaughter, T., Black, M. M. Composite microtubules of the axon: quantitative analysis of tyrosinated and acetylated tubulin along individual axonal microtubules. Journal of Cell Science. 104 (2), 339-352 (1993).
  19. Black, M. M., Slaughter, T., Moshiach, S., Obrocka, M., Fischer, I. Tau is enriched on dynamic microtubules in the distal region of growing axons. Journal of Neuroscience. 16 (11), 3601-3619 (1996).
  20. Caron, J. M., Jones, A. L., Kirschner, M. W. Autoregulation of tubulin synthesis in hepatocytes and fibroblasts. Journal of Cell Biology. 101 (5), 1763-1772 (1985).
  21. Merrick, S. E., Trojanowski, J. Q., Lee, V. M. Selective destruction of stable microtubules and axons by inhibitors of protein serine/threonine phosphatases in cultured human neurons. Journal of Neuroscience. 17 (15), 5726-5737 (1997).
  22. Miyasaka, T., Sato, S., Tatebayashi, Y., Takashima, A. Microtubule destruction induces tau liberation and its subsequent phosphorylation. FEBS Letters. 584 (14), 3227-3232 (2010).
  23. Hagita, A., et al. Quantitative fractionation of tissue microtubules with distinct biochemical properties reflecting their stability and lability. Biochemical and Biophysical Research Communications. 560, 186-191 (2021).
  24. Montecinos-Franjola, F., Chaturvedi, S. K., Schuck, P., Sackett, D. L. All tubulins are not alike: Heterodimer dissociation differs among different biological sources. Journal of Biological Chemistry. 294 (26), 10315-10324 (2019).
  25. Vallee, R. B. A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). Journal of Cell Biology. 92 (2), 435-442 (1982).
  26. Bartolo, M. E., Carter, J. V. Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach. Plant Physiology. 97 (1), 182-187 (1991).
  27. Strang, K. H., Golde, T. E., Giasson, B. I. MAPT mutations, tauopathy, and mechanisms of neurodegeneration. Laboratory Investigation. 99 (7), 912-928 (2019).
  28. Fourel, G., Boscheron, C. Tubulin mutations in neurodevelopmental disorders as a tool to decipher microtubule function. FEBS Letters. 594 (21), 3409-3438 (2020).
  29. Terry, R. D., Gonatas, N. K., Weiss, M. Ultrastructural studies in Alzheimer's presenile dementia. The American Journal of Pathology. 44 (2), 269-297 (1964).
  30. Yoshida, H., Ihara, Y. Tau in paired helical filaments is functionally distinct from fetal tau: assembly incompetence of paired helical filament-tau. Journal of Neurochemistry. 61 (3), 1183-1186 (1993).
  31. Cash, A. D., et al. Microtubule reduction in Alzheimer's disease and aging is independent of tau filament formation. The American Journal of Pathology. 162 (5), 1623-1627 (2003).
  32. Hempen, B., Brion, J. P. Reduction of acetylated alpha-tubulin immunoreactivity in neurofibrillary tangle-bearing neurons in Alzheimer's disease. Journal of Neuropathology and Experimental Neurology. 55 (9), 964-972 (1996).
  33. Miyasaka, T., et al. Imbalanced expression of tau and tubulin induces neuronal dysfunction in C. elegans models of tauopathy. Frontiers in Neuroscience. 12, 415 (2018).
  34. Boiarska, Z., Passarella, D. Microtubule-targeting agents and neurodegeneration. Drug Discovery Today. 26 (2), 604-615 (2021).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Quantitative Microtubule FractionationStable MicrotubulesLabile MicrotubulesFree TubulinMouse TissuesMicrotubule StabilityAlzheimer s DiseaseCancer ResearchProtein Extraction ProtocolTissue HomogenizationCentrifugation TechniquesSDS Sample BufferWestern Blotting Standard Curve

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved