This work was supported by the ANR-10-IDEX-0001-02, the LabEx Cell&#8217;n&#8217;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&#176;2023. JAS was supported by the European Union&#8217;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement No 675737, and the FRM grant FDT201904008210. SB was supported by the FRM grant FDT201805005465.
We thank all members of the Janke lab, in particular J. Souphron, as well as G. Lakisic (Institut MICALIS, AgroParisTech) and A. Gautreau (Ecole Polytechnique) for help during the establishment of the protocol. We would like to thank the animal facility of the Institut Curie for help with mouse breeding and care.
The antibody 12G10, developed by J. Frankel and M. Nelson, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa.

<ol>
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The authors have nothing to disclose.

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...

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 &#8216;tubulin code&#8217;1,2,3. The tubulin code includes two principal components: differential incorporation of &#945;- and &#946;-tubulin gene products (tubulin isotypes) into the microtubules and tubulin posttranslational modifications (PTMs).
Since the 1970s, in vitro reconstitution experiments, combined with evolving light microscopy techniques, have paved the way for important discoveries about the properties of microtubules: dynamic instability4 and treadmilling5, and their other mechanisms and functions6,7,8,9,10,11,12,13,14,15. Almost all the in vitro experiments performed so far have been based on tubulin purified from brain tissue using repeated cycles of polymerization and depolymerization16,17. Although purification from the brain tissue confers the advantage of obtaining high-quality tubulin in large quantities (usually gram amounts), one important drawback is the heterogeneity as tubulin purified from brain tissue is a mixture of different tubulin isotypes and is enriched with many tubulin PTMs. This heterogeneity makes it impossible to delineate the role of a particular tubulin PTM or isotype in the control of microtubule properties and functions. Thus, producing assembly-competent tubulin with controlled tubulin PTMs and homogenous isotype composition is essential to address the molecular mechanisms of the tubulin code.
Recently, an approach to purify tubulin by affinity chromatography using the microtubule-binding TOG (tumor-overexpressed gene) domain of yeast Stu2p has been developed18. In this method, tubulin in crude lysates of cells or tissue is passed through a column where it binds to the matrix-immobilized TOG domain, which allows the analysis of the whole tubulin pool of a given, even very small, sample. A long-awaited approach to purify recombinant tubulin has also been described in recent years. It is based on the baculovirus system, in which a bi-cistronic vector containing &#945;- and&#160;&#946;-tubulin genes is expressed in insect cells19. However, this method is very cumbersome and time-consuming and is therefore mostly used for studying the impact of tubulin mutations20 and tubulin isotypes21,22,23 in vitro.
In the current protocol, we describe a method that uses the well-established and widely used polymerization-depolymerization approach as a blueprint to generate tubulin with different levels of modification either from cell lines or from mouse brain tissue24. In this procedure, tubulin is cycled between the soluble (tubulin dimer at 4 &#176;C) and polymerized form (microtubule at 30 &#176;C in the presence of guanosine 5&#39;-triphosphate [GTP]). Each form is separated through successive steps of centrifugation: tubulin dimers will remain in the supernatant after a cold (4 &#176;C) spin, whereas microtubules will be pelleted at 30 &#176;C. Furthermore, one polymerization step is carried out at high piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES) concentration, which allows the removal of microtubule-associated proteins from the microtubules and thus, from the finally purified tubulin. Tubulin purified from HeLa S3 cells grown as suspension or adherent cultures is virtually free of any tubulin PTM and has been used in recent in vitro reconstitution experiments25,26,27,28. We have further adapted the method to purify tubulin from single mouse brains, which can be used for a large number of mouse models with changes in tubulin isotypes and PTMs.
In the protocol, we first describe the generation of the source material (cell mass or brain tissue), its lysis (Figure 1A), followed by the successive steps of tubulin polymerization and depolymerization to purify the tubulin (Figure 1B). We further describe the process to assess the purity (Figure 2A,B) and quantity (Figure 3A,B) of the purified tubulin. The method can be adapted to produce tubulin enriched with a selected PTM by overexpressing a modifying enzyme in cells prior to tubulin purification (Figure 4B). Alternatively, tubulin-modifying enzymes can be added to tubulin during the purification process. Finally, we can purify tubulin lacking specific isotypes or PTMs from the brains of mice deficient in the corresponding tubulin-modifying enzymes (Figure 4B)29.
The method we describe here has two main advantages: (i) it allows the production of sufficiently large amounts of tubulin in a relatively short time, and (ii) it generates high-quality, pure tubulin, with either specific tubulin isotype composition or PTMs. In the associated video of this manuscript, we highlight some of the critical steps involved in this procedure.

<table><tbody><tr><td>1&amp;nbsp;M MgCl2&amp;nbsp;</td><td>Sigma</td><td>#M1028</td><td></td></tr><tr><td>1-L cell culture vessels</td><td>Techne F7610&amp;nbsp;</td><td></td><td>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&amp;rsquo;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.</td></tr><tr><td>1.5- and 2-ml tubes</td><td></td><td></td><td></td></tr><tr><td>14-ml round-bottom tubes</td><td></td><td></td><td></td></tr><tr><td>15-cm-diameter sterile culture dishes</td><td></td><td></td><td></td></tr><tr><td>15-ml screw-cap tubes</td><td></td><td></td><td></td></tr><tr><td>2-mercaptoethanol&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#M3148</td><td>2-mercaptoethanol is toxic and should be used under the hood.</td></tr><tr><td>4-(2-aminoethyl)-benzenesulfonyl fluoride&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#A8456</td><td></td></tr><tr><td>40% Acrylamide&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>#161-0140</td><td></td></tr><tr><td>5-, 10- 20-ml syringes</td><td></td><td></td><td></td></tr><tr><td>5-ml, 10-ml, 25-ml sterile pipettes</td><td></td><td></td><td></td></tr><tr><td>50-ml screw-cap tubes</td><td></td><td></td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Sigma</td><td>#A3678</td><td></td></tr><tr><td>Anti-alpha-tubulin antibody, 12G10&amp;nbsp;</td><td>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</td><td></td><td>dilution: 1/500</td></tr><tr><td>Anti-glutamylated tubulin antibody, GT335&amp;nbsp;</td><td>AdipoGen&amp;nbsp;</td><td>#AG-20B-0020</td><td>dilution: 1/20,000</td></tr><tr><td>Aprotinin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#A1153</td><td></td></tr><tr><td>Balance (0.1 &amp;ndash; 10 g)</td><td></td><td></td><td></td></tr><tr><td>Beckman 1-l polypropylene bottles&amp;nbsp;</td><td></td><td></td><td>For collecting spinner cultures</td></tr><tr><td>Beckman Avanti J-26 XP centrifuge</td><td></td><td></td><td>For collecting spinner cultures</td></tr><tr><td>Biological stirrer&amp;nbsp;</td><td>Techne MCS-104L&amp;nbsp;</td><td></td><td>Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells</td></tr><tr><td>Bis N,N&amp;rsquo;-Methylene-Bis-Acrylamide&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>#161-0201</td><td></td></tr><tr><td>Blender IKA Ultra-Turrax&amp;reg;&amp;nbsp;</td><td></td><td></td><td>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.</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma&amp;nbsp;</td><td>#A7906</td><td></td></tr><tr><td>Bromophenol blue&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#1.08122</td><td></td></tr><tr><td>Carboxypeptidase A (CPA)</td><td>Sigma</td><td>#C9268</td><td>Concentration: 1.7&amp;nbsp;U/&amp;micro;l</td></tr><tr><td>Cell culture hood</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator set at 37&amp;deg;C, 5% CO2</td><td></td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>&amp;nbsp;Sigma&amp;nbsp;</td><td>#D8418</td><td>DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection.</td></tr><tr><td>DMEM medium&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#41965062</td><td></td></tr><tr><td>DTT, DL-Dithiothreitol&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#D9779</td><td></td></tr><tr><td>EDTA</td><td>Euromedex</td><td>#EU0007-C</td><td></td></tr><tr><td>EGTA</td><td>Sigma&amp;nbsp;</td><td>#E3889</td><td></td></tr><tr><td>Ethanol absolute&amp;nbsp;</td><td>Fisher Chemical&amp;nbsp;</td><td>#E/0650DF/15</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma&amp;nbsp;</td><td>#F7524</td><td></td></tr><tr><td>French pressure cell press&amp;nbsp;</td><td>Thermo electron corporation</td><td>&amp;nbsp;#FA-078A</td><td>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).</td></tr><tr><td>Glycerol</td><td>&amp;nbsp;VWR Chemicals&amp;nbsp;</td><td>#24388.295</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>#G8898</td><td></td></tr><tr><td>GTP&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#G8877</td><td></td></tr><tr><td>Heating block&amp;nbsp;</td><td>Stuart&amp;nbsp;</td><td>#SBH130D</td><td></td></tr><tr><td>Hela cells&amp;nbsp;</td><td></td><td>ATCC&amp;reg; CCL-2&amp;trade;</td><td></td></tr><tr><td>Hela S3 cells&amp;nbsp;</td><td>ATCC</td><td>ATCC&amp;reg; CCL-2.2&amp;trade;</td><td></td></tr><tr><td>Hydrochloric acid (HCl )</td><td>VWR</td><td>#20252.290</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td></td><td></td><td>With fluorescence if cell transfection is to be verified</td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>#20842.298</td><td></td></tr><tr><td>jetPEI</td><td>Polyplus&amp;nbsp;</td><td>#101</td><td></td></tr><tr><td>JLA-8.1000 rotor&amp;nbsp;</td><td></td><td></td><td>For collecting spinner cultures</td></tr><tr><td>KOH&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#P1767</td><td>KOH is corrosive and causes burns; use eye and skin protection.</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#25030123</td><td></td></tr><tr><td>Laboratory centrifuge for 50-ml tubes</td><td>Sigma</td><td>4-16 K&amp;nbsp;</td><td></td></tr><tr><td>Leupeptin&amp;nbsp;</td><td>Sigma</td><td>#L2884</td><td></td></tr><tr><td>Liquid nitrogen&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips</td><td></td><td></td><td></td></tr><tr><td>Micropestles</td><td>Eppendorf</td><td>#0030 120.973</td><td></td></tr><tr><td>Mouse brain tissue&amp;nbsp;</td><td></td><td></td><td>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.</td></tr><tr><td>Needles 18G X 1 &amp;frac12;&amp;rdquo; (1.2 X 38 mm</td><td>Terumo</td><td>#18G</td><td></td></tr><tr><td>Needles 20G X 1 &amp;frac12;&amp;rdquo; (0.9 X 38 mm</td><td>Terumo</td><td>#20G</td><td></td></tr><tr><td>Needles 21G X 4 &amp;frac34;&amp;rdquo; (0.8 X 120 mm</td><td>B.Braun</td><td>#466 5643</td><td></td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#14190169</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#15140130</td><td></td></tr><tr><td>pH-meter</td><td></td><td></td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>Sigma&amp;nbsp;</td><td>#P7626</td><td>PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions.</td></tr><tr><td>PIPES&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#P6757</td><td></td></tr><tr><td>Pipette-boy</td><td></td><td></td><td></td></tr><tr><td>Rotors</td><td>Beckman 70.1 Ti; TLA-100.3; and TLA 55</td><td></td><td></td></tr><tr><td>SDS-PAGE electrophoresis equipment&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>#1658001FC</td><td></td></tr><tr><td>SDS, Sodium dodecyl sulphate&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>#442444H</td><td>For preparing Laemmeli buffer&amp;nbsp;</td></tr><tr><td>SDS, Sodium dodecyl sulphate&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#L5750</td><td>For preparing &#039;TUB&#039; SDS-PAGE gels</td></tr><tr><td>Sonicator&amp;nbsp;</td><td>Branson</td><td>#101-148-070</td><td>Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at &amp;ldquo;Output control&amp;rdquo; 1, &amp;ldquo;Duty cycle&amp;rdquo; 10% and time depending on the cell type used.</td></tr><tr><td>Tabletop centrifuge for 1.5 ml tubes</td><td>Eppendorf</td><td>5417R&amp;nbsp;</td><td></td></tr><tr><td>TEMED, N, N, N&amp;prime;, N&amp;prime;-Tetramethylethylenediamine&amp;nbsp;</td><td>Sigma</td><td>#9281</td><td></td></tr><tr><td>Trichostatin A (TSA)</td><td>Sigma</td><td>#T8552</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#T9284</td><td></td></tr><tr><td>Trizma base (Tris)</td><td>Sigma&amp;nbsp;</td><td>#T1503</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Life Technologies</td><td>#15090046</td><td></td></tr><tr><td>Ultracentrifuge rotors&amp;nbsp;</td><td></td><td>TLA-55, TLA-100.3 and 70.1 Ti rotors</td><td>Set at 4&amp;deg;C or 30&amp;deg;C based on the need of the experiment&amp;nbsp;</td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman</td><td>#357448&amp;nbsp;</td><td>for using with TLA-55 rotor</td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman</td><td>#349622</td><td>for using with TLA-100.3 rotor</td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman</td><td>#355631&amp;nbsp;</td><td>for using with 70.1 Ti rotor</td></tr><tr><td>Ultracentrifuges</td><td>Beckman</td><td>Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent)</td><td>Set at 4&amp;deg;C or 30&amp;deg;C based on the need of the experiment&amp;nbsp;</td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>Water bath equipped with floaters or tube holders</td><td></td><td></td><td>Set at 30&amp;deg;C&amp;nbsp;</td></tr></tbody></table>

purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles

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 &#8220;tubulin code&#8221; 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.

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<...

Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

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

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 ...

purification-tubulin-with-controlled-posttranslational-modifications

Research

JoVE Journal

Biochemistry

5.1K Views.  Institut Curie. Dit protocol beschrijft tubulinezuivering uit kleine/middelgrote bronnen zoals gekweekte cellen of enkele muizenhersenen, met behulp van polymerisatie- en depolymerisatiecycli. De gezuiverde tubuline is verrijkt in specifieke isotypes of heeft specifieke posttranslationele modificaties en kan worden gebruikt in in vitro reconstitutietesten om microtubulidynamiek en interacties te bestuderen.

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

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4&#8211;8 &#181;m/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.

The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method ...

Engineering

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 networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

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

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

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

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

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 especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

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

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the posttranslational modifications, microtubules can form complexes with various interacting proteins. These microtubule-protein complexes are often implicated in human diseases. Understanding the structure of such complexes is useful for elucidating their mechanisms of action and can be studied by cryo-electron microscopy (cryo-EM). To obtain such complexes for structural studies, it is important to extract microtubules containing or lacking specific posttranslational modifications. Here, we describe a simplified protocol to extract endogenous tubulin from genetically modified mammalian cells, involving microtubule polymerization, followed by sedimentation using ultracentrifugation. The extracted tubulin can then be used to prepare cryo-electron microscope grids with microtubules that are bound to a purified microtubule-binding protein of interest. As an example, we demonstrate the extraction of fully tyrosinated microtubules from cell lines engineered to lack the three known tubulin-detyrosinating enzymes. These microtubules are then used to make a protein complex with enzymatically inactive microtubule-associated tubulin detyrosinase on cryo-EM grids.

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...

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.

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 ...

Efficient Tubulin Isolation from Neural Tissue

Optimizing Tubulin Yield from Porcine Brain Tissue

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we present a procedure for extracting tubulin that can be employed, with minor modifications, for neural tissue from multiple sources. In the presented protocol, a new clarification step of the crude lysate has been introduced, which led to a significant reduction in the initial amount of insoluble debris before the first polymerization step occurred. This additional step allowed the processing of additional tissue while using the same instrumentation and, thus, increasing the relative volume of the processed homogenate. The newly introduced step has no significant effect on the quality of purified tubulin, as confirmed by in vitro activity assays and transmission electron microscopy. The described procedure contains all crucial steps, including tissue collection, transport, tissue homogenization, tubulin isolation cycles, and final polishing by ion exchange chromatography using FPLC and subsequent activity measurement assays. The homogeneity of purified tubulin was more than 97%, as confirmed by MS/MS analysis utilizing electrospray ionization and MALDI-TOF.

Author Spotlight: Purifying High-Quality Tubulin to Study Protein Dynamics and Therapeutic Applications

This protocol describes a technique for the high-yield isolation of tubulin from the porcine brain optimized for small-scale instrumentation. The isolation procedures are complemented by procedures for determining tubulin polymerization activity in vitro using co-sedimentation assays and transmission electron microscopy.

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we ...

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

Biology

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

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

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