Name: quantitative microtubule fractionation technique to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues
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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 &#34;Brain Protein Aging and Dementia Control&#34; from MEXT (TM; 26117004), and by Uehara Research Fellowship from the Uehara Memorial Foundation (TM; 202020027). The authors declare no competing financial interests.

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....

<ol>
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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...

Microtubules (MTs) are elongated tubular structures comprising protofilaments consisting of &#945;/&#946;-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 &#945;-tubulin subunit is exposed, is relatively stable, whereas the plus-end, where the &#946;-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&#160;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 &#945;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 ML TUBE CASE OF 500</td>
			<td>Beckman Coulter</td>
			<td>357448</td>
			<td></td>
		</tr>
		<tr>
			<td>1A2</td>
			<td>Sigma-Aldrich</td>
			<td>T9028</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td>Nacalai Tesque</td>
			<td>02442-44</td>
			<td></td>
		</tr>
		<tr>
			<td>300 kDa ultrafiltration spin column</td>
			<td>Aproscience</td>
			<td>PT-1013</td>
			<td></td>
		</tr>
		<tr>
			<td>6-11B1</td>
			<td>Sigma-Aldrich</td>
			<td>T7451</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>AKTA prime plus</td>
			<td>Cytiva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-035-146</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>antipain</td>
			<td>Peptide Institute Inc.</td>
			<td>4062</td>
			<td></td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Nacalai Tesque</td>
			<td>03346-84</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemi-Lumi One L</td>
			<td>Nacalai Tesque</td>
			<td>07880-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filter system</td>
			<td>Corning</td>
			<td>430758</td>
			<td>0.22&#181;m 33.2cm&#178; Nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>DIFP</td>
			<td>Sigma-Aldrich</td>
			<td>55-91-4&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DIGITAL HOMOGENIZER</td>
			<td></td>
			<td>AS ONE</td>
			<td>HOM</td>
		</tr>
		<tr>
			<td>DM1A</td>
			<td>Sigma-Aldrich</td>
			<td>T9026</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Nacalai Tesque</td>
			<td>14128-46</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Nacalai Tesque</td>
			<td>37346-05</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroTrans W 3.3 Meter Roll</td>
			<td>Pall Corporation</td>
			<td>BSP0161</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Nacalai Tesque</td>
			<td>17018-25</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td></td>
			<td>Nacalai Tesque</td>
			<td>17450-61</td>
		</tr>
		<tr>
			<td>HIGH SPEED REFRIGERATIOED MICRO CENTRIFUGE Kitman</td>
			<td></td>
			<td>TOMY</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/600 Superdex 200 pg column</td>
			<td>Cytiva</td>
			<td>28-9893-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Gauge Software&#160;</td>
			<td>FUJIFILUM Wako Pure Chemical Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoStar LD&#160;</td>
			<td>FUJIFILUM Wako Pure Chemical Corporation</td>
			<td>292-69903</td>
			<td></td>
		</tr>
		<tr>
			<td>KMX-1</td>
			<td>Millipore</td>
			<td>MAB3408</td>
			<td>1:5,000 dilution</td>
		</tr>
		<tr>
			<td>LAS-4000 luminescent image analyzer</td>
			<td>FUJIFILUM Wako Pure Chemical Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Peptide Institute Inc.</td>
			<td>43449-62</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Nacalai Tesque</td>
			<td>21003-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Nacalai Tesque</td>
			<td>32013-92</td>
			<td></td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Nacalai Tesque</td>
			<td>31420-82</td>
			<td></td>
		</tr>
		<tr>
			<td>okadaic acid</td>
			<td>LC Laboratories</td>
			<td>O-2220&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTIMA MAX-XP</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Nacalai Tesque</td>
			<td>26436-52</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Nacalai Tesque</td>
			<td>27327-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Centrifuge Tubes for TLA120.2</td>
			<td>Beckman Coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (cOmplete, EDTA-free)</td>
			<td></td>
			<td>Roche</td>
			<td>5056489001</td>
		</tr>
		<tr>
			<td>Purified tubulin&#160;</td>
			<td>Cytoskeleton</td>
			<td>T240</td>
			<td></td>
		</tr>
		<tr>
			<td>QSONICA Q55</td>
			<td>QSonica</td>
			<td>Q55</td>
			<td></td>
		</tr>
		<tr>
			<td>Taxol</td>
			<td></td>
			<td>LC Laboratories</td>
			<td>P-9600</td>
		</tr>
		<tr>
			<td>TLA-120.2 rotor</td>
			<td>Beckman Coulter</td>
			<td>357656</td>
			<td></td>
		</tr>
		<tr>
			<td>TLA-55 rotor</td>
			<td>Beckman Coulter</td>
			<td>366725</td>
			<td></td>
		</tr>
		<tr>
			<td>TLCK</td>
			<td>Nacalai Tesque</td>
			<td>34219-94</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai Tesque</td>
			<td>12967-45</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative microtubule fractionation technique to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues

Microtubules, composed of &#945;/&#946;-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 &#215; g) to remove debris. The first ultracentrifugation step (100,000 &#215; g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 &#215; 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.

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 &#215; g for 20 min accounted for 34.86% &#177; 1.68% of total tubulin in a mouse brain. The supernatant (S2) was fur...

Watch this Scientific Journal Video about Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues at JoVE.com

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

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.

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit ...

Our new protocol helps to distinguish and quantify three statuses of tube rings, such as stable microtubules, labile microtubules, and free tubules in animal tissues. This technique is composed of simple steps and can evaluate the structural stability of tubing, which cannot be detected by quantification of tubing post translational modification. This method is essential for the research and development of therapeutic ways for diseases where microtubule stability is critical, such as Alzheimer's disease and cancers.Start the procedure by preparing the lab wear for tissue dissection. Fill a box with crushed ice and place two Petri dishes on it. Fill one dish with ice cold phosphate buffer solution or PBS for transient wash and storage of dissected tissues.Lay filter paper moistened with PBS on the second dish. Next, weigh the 1.5-milliliter microtubes filled with PBS for dissected tissue storage. After extracting the tissues from a euthanized mouse, transfer them to the tubes and re-weigh each microtube.Move the tissues into a glass homogenizer with ice-cold microtubule stabilizing buffer or MSB+medium, and homogenize the tissue immediately using a chilled homogenizer. Now transfer the tissue homogenate to a two-milliliter microtube using a Pasteur pipette and centrifuge at 2, 400 G for three minutes at two degrees Celsius. Transfer the supernatant to a new 1.5-milliliter microtube to remove the debris.Vortex the supernatant or the S1 fraction in a new 1.5-milliliter microtube. Immediately pipette 200 microliters of the S1 fraction into a centrifugation microtube and centrifuge at 100, 000 G using a TLA-55 rotor for 20 minutes at two degrees celsius. After the second centrifugation, separate the S2 supernatant from the P2 precipitate.Immediately transfer the whole amount of the S2 fraction into a centrifugation microtube and use a TLA-120.2 rotor to spin it at 500, 000 G for 60 minutes at two degrees Celsius. After the third centrifugation, separate the S3 supernatant from the P3 precipitate. Dissolve the entire S3 fraction in 200 microliters of 2X sodium dodecyl sulfate, or SDS sample buffer.Mix the remaining S1 fractions with an equal volume of 2X SDS sample buffer for use as a standard curve for western blotting. Next, add 400 microliters of SDS sample buffer to the P2 and P3 fraction tubes. Then, briefly sonicate the solution to dissolve the precipitate before transferring the samples into new 1.5-milliliter microtubes and placing them in the icebox.Boil all the samples at 100 degrees Celsius for three minutes. Microtubules in the P2 fraction accounted for 34.86%of the total alpha-tubulin in a mouse brain, whereas those in the P3 and S3 fractions accounted for 56.13%and 9.01%respectively. The percentage of beta-3 tubulin in the P2, P3, and S3 fractions were not significantly different from those of alpha-tubulin.The S2 fraction showed limited passage of tubulin through a 300-kilodalton ultrafiltration spin column, while the S3 fraction allowed for complete passage of tubulin complexes. Chromatographic separation resulted in a 100-kilodalton elution of S3 tubulin. Equal proportions of alpha-and beta-tubulin were recovered in each fraction.P2 fractions were significantly enriched with acetylated alpha-tubulin, While tyrosinated alpha-tubulin was dominant in the P3 fraction. Freezing the brain prior to homogenization resulted in decreased alpha-tubulin concentration in the P2 fractions. However, alpha-tubulin increased in the P3 fraction.Freezing also decreased the acetylation levels and increased tyrosination levels of alpha-tubulin in the P2 fraction. Nocodazole treatment decreased alpha-tubulin in the P2 fraction. In contrast to freezing, nocodazole did not affect the P2 tubulin post-translational modifications.The brain tissue exhibited a significantly higher concentration of P2 tubulins, whereas P3 tubulins were concentrated in proliferative tissue. Western blotting showed that the P2 tubulin specifically found in the nervous system originated from stable microtubules, and P3 tubulin originated from labile microtubules. Microtubules stability is highly dynamic.It is important to complete this protocol quickly, without interruptions, in the cold temperature environment. Also ensure that the tissues and fractions have not been frozen until being dissolved in SDS sample buffer. Combining immune presentation using each fraction with proteomics is expected to identify factors involved in microtubule dynamics.Using this method, we revealed how normal tau exists on microtubules. It can also be used to study how it becomes abnormal in aged brains in order to identify new drug targets of dementia.

Research

JoVE Journal

Biochemistry

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...