The authors would like to thank all the summer 2021 Ross Lab members, especially K. Alice Lindsay, for their help. This work was supported by a grant from NSF BIO-2134215 that supported S. Sahu, N. Goodbee, H.B. Lee, and J.L. Ross. A grant from the KECK Foundation (Rae Anderson, USD, lead PI) partially supported R. Branch and P. Chauhan&#160;

<ol>
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The authors declare that they have no competing financial interests.

The methods described here have been used in several papers to create microtubule tactoids (Figure 2)22,28. These experiments are biologically relevant to help uncover the organizational principles that control the shape and stability of the mitotic or meiotic spindle in most cell types. In addition, microtubules are model liquid crystal mesogens that can help in learning more about how liquid crystals nucleate and grow nematic phases from isotropic phases.
The procedure outlined here has several advantages for exploring microtubule self-organization. First, it is highly reproducible, having been performed in the lab by many students, including high school students, with little foreknowledge or training before starting in the lab. The tactoids are birefringent22, allowing them to be viewed in transmitted light in addition to fluorescence microscopy, making this method accessible to many labs and this experimental procedure adaptable to educational purposes, in addition to high-end research. Finally, this process opens avenues to continue to understand and probe biological systems in a stripped-down, reductionist approach, allowing one to understand how each additional condition, protein, or additive can alter the self-organization of tactoids and, perhaps, ultimately, the spindle. Targets for better biomimicry include activity, fluidity, and filament polarity sorting.
There can be several factors affecting the experiment giving unexpected results. For example, if the tactoids do not form (Figure 2) but fan-like patterns are observed, the MAP65 is likely not present or not binding to the microtubules22,28. This should be obvious in the MAP65 fluorescence channel as well because the GFP-MAP65 will not be bound to the microtubules.
If the tactoids do not form and the background appears as speckles on the glass, this could be due to the surface coating. Once performed, the silanization lasts only 1 month on coverslips. When it wears off, the tubulin will be able to bind to the exposed surface non-specifically. This binding will occur in odd patterns.
If the tactoids do not form and the tubulin is observed in aggregates of various shapes and sizes, this could be due to poor-quality tubulin. Tubulin can be centrifuged to remove initial aggregates that can drive this off-pathway aggregation instead of microtubule polymerization. If the surface is binding to the tubulin, it can also deplete the tubulin in the solution. Low concentrations of tubulin, below the critical concentration for polymerizing microtubules, can result in aggregates.
In FRAP experiments, if the MAP65 channel does not show any recovery (Figure 5), it is possible that the photobleaching was photodamaging the microtubules. Photodamage causes the localized destruction of the filaments. This can be checked by examination in the transmitted channel. Microtubule tactoids are visible in the transmitted channel through a high index of refraction mismatch with the surrounding water. Light-induced photodamage will appear as a burn mark or loss of contrast in transmitted light imaging at the location of the ROI subjected to photobleaching. If this occurs, the laser or light power must be reduced to inhibit the photodamage of the proteins.
There were several challenges encountered in this procedure and approach. One issue is that the length measures are currently performed by hand by clicking on the image. This method, while straightforward, can result in high uncertainty. The width measurement that uses the cross-section and fitting to a Gaussian is a better method for quantifying the size. A similar method could be employed for the length. A second issue is that, sometimes, the tactoids, because they are so long and thin, can bend. This makes quantifying the length more difficult. The contour length can be quantified using a segmented line, but there is added uncertainty each time a segment is added.
From a scientific perspective, this approach has some other challenges for its use as a model for liquid crystals or spindles. The first challenge has been the long, thin shape of the tactoids that microtubules create (Figure 3 and Figure 4). As noted in prior publications22, microtubule tactoids are homogeneous tactoids, not bipolar. This means that the microtubules that make up the shape do not reorient to point toward the tips of the structure. Instead, all the microtubules are parallel to the long axis and the &#34;poles&#34; are located at infinity. This is very different from the tactoids observed for molecular liquid crystals or even for actin or DNA that can also act as liquid crystal mesogens. In these other systems, the tactoids are bipolar and, when viewed in crossed polarizers, they show the telltale signs of reorientation of the rods.
A second major challenge in this system is that the microtubules are immobile inside the tactoid. This is clear from FRAP experiments and analysis as the recovery of the microtubules is very low. Their solid-like nature makes microtubule tactoids less valuable as large-scale liquid crystal analogs. The nematic phase of a liquid crystal should have both liquid (fluid) and crystal (organized) properties. Although the shape seems right for the spindle, the immobility makes the system less exciting as a model mitotic spindle. On the other hand, this issue provides opportunities to investigate how one can modify the experiments to create more fluidity in the system.
These scientific challenges offer exciting opportunities that will allow new knowledge about the system. To make the microtubule tactoids more bipolar, one could use shorter microtubules. There is an added challenge, however, since microtubules do not have many well-characterized capping proteins to control the length as actin does. The use of nucleation and growth requires the use of very high concentrations of tubulin and GMPCPP to make short microtubules. The high tubulin concentration results in a larger number of filaments in the system, which makes it more difficult to separate tactoids from one another. The addition of new microtubule cappers, such as DARPin33, may help with this situation. The second issue of the microtubules being immobile could be mitigated by the addition of motor proteins, such as kinesin-534, which are tetramers of motors used in mitosis. Alternatively, artificial dimers of dimeric kinesin-1 could be used15.
Another way to add more fluidity would be to allow the microtubules to perform their dynamic instability, the growing and shrinking of microtubules. Currently, microtubules that are seeded with stable GMPCPP filaments and then undergo dynamic instability are far longer than desired to form a spindle or tactoid, which would result in very long organizations like fans or bundles. So, adding microtubule dynamic instability would need to be done carefully to preserve the tactoid shape. The addition of associated proteins and enzymes that can control the length may mitigate this issue. For instance, depolymerizing kinesins, such as kinesin-1335, or severing enzymes, like katanin36, would likely be needed. These experiments are complex and difficult, although they would be very insightful no matter what the results reveal. Whichever direction future experiments take, the platform developed here for creating microtubule tactoids can expose new information on the physical basis of microtubule organization.

Cell division via mitosis is one of the most important biological processes to sustain life. The microtubule filaments, composed of tubulin dimers, are essential structural elements of this process. The transient machinery created in metaphase when the chromosomes are aligning in the cell center is called the mitotic spindle due to its shape, which is like a spindle of a loom covered in threads (Figure 1A). It is well-established across many organisms that microtubules are used in metaphase to push and pull condensed chromosomes into the center of the cell, aligning them and hooking them up to microtubules that will pull them apart in anaphase (Figure 1B,C). The spindle forms in both meiosis (Figure 1B) and mitosis (Figure 1C), created from many overlapping microtubules not wrapped around the central axis like thread but running parallel to the interface. Creating these microtubule-based structures requires associated proteins that crosslink and associated enzymes that can act as motors to help push and pull the chromosomes1.
Studies of meiotic spindles have shown that the microtubules are short, dynamic, and overlapping in crosslinked arrays2,3,4,5,6 (Figure 1Di). Due to the physical organization of these short microtubules, the meiotic spindle is similar to a liquid crystal tactoid (Figure 1E). Indeed, spindles have been shown to coalesce and merge, as one would expect from liquid crystal tactoids5.
Many studies dating back to the 1960s have used fixation, serial sections, and electron microscopy to determine that there are two types of microtubules inside the mitotic spindle7,8,9,10. The first type is called the kinetochore microtubules, which connect the spindle pole to the kinetochore. The second type is called the interpolar or polar microtubules, which grow past the chromosomes and overlap at the midzone (Figure 1Dii)8,9,10. A third type is called the astral microtubules, which are outside of the spindle and connect the poles to the cell edge; these microtubule organizations are outside of the scope of the current discussion. There have been recent studies on the interaction between augmin6 and gamma-tubulin ring complex influencing nucleation centers for microtubules,&#160;resulting in a mitotic spindle with shorter microtubules like in Figure 1D.
Since microtubules are longer than they are wide, with a high aspect ratio and high stiffness, they are like scaled-up versions of liquid crystal molecules. In soft matter physics, atoms and molecules have been approximated using minimal interactions to deduce the physical mechanisms of phase transitions, including nucleation and the melting of crystals11.&#160;Similarly, microtubules are mesoscale objects that are scaled-up versions of liquid crystal molecules, giving insights into the physics of liquid crystal dynamics, including the nucleation and growth of the nematic phases from isotropic ones. Further, as discussed above, the meiotic spindle displays properties like those of a liquid crystal tactoid, a nematic state that nucleates and grows from the isotropic state of liquid crystal molecules3,4,5. For tactoids, nucleation and growth are like that of other crystals (i.e., requiring a relatively high concentration of mesogens [the molecules that form liquid crystals]). The unique &#34;spindle&#34; shape of the tactoid comes from the local alignment of the liquid crystal mesogens that align into the nematic phase (Figure 1E). They cannot form a rounded crystal because the molecules are highly asymmetric. Given the nature of the microtubules, it is perhaps not surprising that the mitotic spindle-machinery made from a high local concentration of microtubules is also of the same shape, whether it is called a tactoid or spindle. Tactoids can be bipolar, with poles at the tapered ends (Figure 1Ei), or homogeneous, with poles effectively at infinity (Figure 1Eii).
Given the importance of spindle formation, efforts have been underway toward self-organized spindle formation in vitro by demonstrating microtubule condensation into bundles via ionic species12,13, crowding agents that create depletion interactions14,15, and specific microtubule crosslinking proteins13,16,17,18,19,21. Surprisingly, although these agents all work to increase the local concentration of microtubules, they often result in long microtubule bundles but not tactoids. One reason why these bundles are long could be that the microtubules comprising them are also long. Recent work using shorter microtubules also reported longer bundles that are not tapered at the end15; in this case, the bundles are held together with motor proteins that cause extension of the bundles and, thereby, make them longer. Short microtubules with non-extensile crosslinkers are needed for tapered, spindle-like assemblies, as described here.
Recently, we have developed a technique to enable the creation of microtubule tactoids using the antiparallel crosslinker, MAP65, in the presence of nucleating stable microtubules22. The microtubules needed to be short, yet few known regulators of microtubule length can cap microtubules against dynamic instability or end-to-end annealing. Instead, GMPCPP was used to nucleate and stabilize the filaments after growth. This allowed for creating a high density of short microtubules that could self-organize into tactoids. These tactoids were homogeneous when viewed under birefringence. In addition to short microtubules, a specific antiparallel crosslinker, MAP65, was employed to form the tactoids (Figure 2). MAP65 is a plant microtubule-associated protein in the PRC1/Ase1 family of mitotic crosslinkers23. MAP65 exists as a dimer, with a strong affinity to bind to itself as well as the microtubules24. Unlike the meiotic spindle and tactoids observed with actin filaments25,26,27, which are bipolar and have the liquid-like properties of liquid crystals, microtubule tactoids have been observed to be solid-like22,28.
Here, protocols are presented for creating the microtubule tactoids and characterizing the shape of the assemblies and the mobility of the constituents using fluorescence-based techniques.

<table><tbody><tr><td>2% Dichlorodimethylsilane</td><td>GE Healthcare</td><td>118945</td><td>A hydrophobic silane surface treatment. This can be resused upto three times and kept at room temperature for one year.</td></tr><tr><td>5-minute epoxy</td><td>Bob Smith Industries</td><td></td><td>For sealing experimental chambers</td></tr><tr><td>Acetone</td><td>Fisher</td><td>32900HPLC</td><td>100%</td></tr><tr><td>BL21 cells</td><td>Bio Labs</td><td>C2527I</td><td>Competent bacterial cells used to express MAP65</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C30-500MG</td><td>Part of the oxygen scavenging system. A 300 mg/ml stock is made in ddH2O and stored in 10 &amp;mu;l aliquots in -20&amp;deg;C for upto one year.</td></tr><tr><td>Coverslips (22 mm x 22 mm or 22 mm x 30 mm)</td><td>Fisher</td><td>12544-AP</td><td>For experimental chambers</td></tr><tr><td>Dithiothreitol</td><td>Sigma</td><td>43815-5G</td><td>A small molecule that is used to break disulfide bonds and scavenge oxygen. A 1 M stock is made from powder (Sigma) in ddH2O. The solution is aliquoted and stored at -20C for upto one year. The aliquots are used 7-8 times then discarded.</td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889-10G</td><td>Tubulin buffer base ingredient</td></tr><tr><td>Eppendorf 0.5 ml tubes</td><td>Eppendorf</td><td>05 402 18</td><td>For holding experimental solutions</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>111000200</td><td>200 proof</td></tr><tr><td>Filter paper</td><td>Whatman</td><td>1004-110</td><td>For cleaning and for pulling solutions through experimental chambers</td></tr><tr><td>Fluorescence microscope with high NA objectives</td><td>Nikon</td><td>Ti-E, W2 Confocal</td><td>For imaging experiments</td></tr><tr><td>Freezer -20&amp;deg;C</td><td>Fisherbrand</td><td>13986148</td><td>For storing reagents</td></tr><tr><td>Freezer -80&amp;deg;C</td><td>Thermo scientific</td><td>328223H01-C</td><td>For storing proteins.</td></tr><tr><td>Glass containers for silanization</td><td>Michaels</td><td></td><td>For preparing experimental chambers - treating cover glasses</td></tr><tr><td>Glass slides</td><td>Fisher</td><td>12544-4</td><td>For experimental chambers</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7528-250G</td><td>Part of the oxygen scavenging system. A 300 mg/ml stock is made in ddH2O and stored in 10 &amp;mu;l aliquots in -20&amp;deg;C.</td></tr><tr><td>Glucose oxidase</td><td>Sigma</td><td>G2133-250KU</td><td>Part of the oxygen scavenging system. This is stored at 4&amp;deg;C for upto one year.</td></tr><tr><td>GMPCPP</td><td>Jenna Bioscience</td><td>NU-4055</td><td>Slowly hydrolyzable analog of GTP used to polymerize and stabilize the microtubules by reducing the dynamic instability and spontaneous critical concentration for microtubule nucleation to get a consistent length. We purchase a 10 mM stock from Jena Biosciences and store it at -20&amp;deg;C for upto one year.</td></tr><tr><td>Heating element for microscope</td><td>Okolab stage top incubator</td><td></td><td>For imaging experiments</td></tr><tr><td>Imidizole</td><td>Sigma</td><td>I2399</td><td>Used to elute MAP65 protein from Nickel beads to purify MAP65</td></tr><tr><td>Kimwipes</td><td>Fisher</td><td>34155</td><td>For cleaning and for pulling solutions through experimental chambers.</td></tr><tr><td>KOH</td><td>Sigma</td><td>P250-500</td><td>1 M in ddH2O, made fresh to prevent acidification over time.</td></tr><tr><td>Liquid Nitrogen</td><td>Airgas</td><td>NI 230LT22</td><td>To drop freeze aliquots. This can also be used for storage (not recommended)</td></tr><tr><td>MAP65 protein</td><td>Ram Dixit</td><td></td><td>A microtubule-associated protein (MAP) with a molecular weight of 65 kD that is an antiparallel microtubule crosslinker. We have purified an unlabeled and GFP-labeled version of MAP65-1 from Arabidopsis thaliana. We mix the GFP-MAP65 with the unlabeled MAP65 such that 10% of the protein is labeled. The working stock is a 10.8 &amp;mu;M solution. This working solution is stored at 4&amp;deg;C and remade fresh every week. The protocol for purifying the MAP65 and GFP-MAP65 is given in our prior methods chapter (26).</td></tr><tr><td>MgSO4</td><td>Sigma</td><td>MKCJ940</td><td>Tubulin buffer ingredient</td></tr><tr><td>NTA-Nickel beads</td><td>Qiagen</td><td>30210</td><td>Beads required to purify 6xHis tagged MAP65 proteins</td></tr><tr><td>Optomicroscan</td><td>Nikon</td><td></td><td>405 nm laser system which can focus the laser in any desired shape of region of interest</td></tr><tr><td>PEM80</td><td></td><td></td><td>Neutral tubulin polymerizing base buffer for solution made from 80 mM K-PIPES, pH 6.8, 1 mM MgSO4, 1 mM EGTA, stored at 4&amp;deg;C for upto one year.</td></tr><tr><td>Permanent double-sided tape</td><td>3M - Scotch</td><td>34-8724-5691-7</td><td>For experimental chambers</td></tr><tr><td>Petri dish</td><td>Fisher</td><td>FB0875713</td><td>For humid chamber</td></tr><tr><td>PIPES</td><td>Sigma</td><td>P7643-100G</td><td>Tubulin buffer base ingredient</td></tr><tr><td>Pluronic-F127</td><td>Sigma</td><td>P2443-250G</td><td>A block-copolymer with two hydrophilic polyethylene oxide (PEO) blocks on the ends and a hydrophobic center block of polyphenylene oxide (PPO) hydrophobic surface coating we use to prevent protein binding to the surface. Pluronic-F127 is purchased as a powder (Sigma) and dissolved to a 5% (w/v) solution in ddH2O overnight. Once dissolved, the solution can be stored at room temperature for upto one year.</td></tr><tr><td>Polyethylene Glycol</td><td>Affymetrix Inc</td><td>19966 500 GM</td><td>A crowding agent used to create depletion forces to bring tactoids to the surface and help to organize microtubules. We create a 5% (w/v) solution of 100 kDa PEG in PEM80. The solution is stored in 4&amp;deg;C for upto one year and placed on the rocker before use. It is viscous, so we recommend using a positive displacement pipette to work with it.</td></tr><tr><td>Positive displacement pipette</td><td>Eppendorf</td><td></td><td>For pipetting viscous liquids</td></tr><tr><td>Racks for coverslips</td><td>Electron Microscopy Sciences</td><td>72240</td><td>For preparing experimental chambers - treating cover glasses</td></tr><tr><td>Refrigerator 4&amp;deg;C</td><td>Fisherbrand</td><td></td><td>For storing reagents</td></tr><tr><td>Tubulin Labeled</td><td>Cytoskeleton</td><td>TL590M</td><td>lyophilized rhodamine-labeled tubulin from pig brain is purchased (Cytoskeleton) and stored in -80&amp;deg;C until hydrated in PEM80 and used for upto one year.</td></tr><tr><td>Tubulin protein</td><td>Cytoskeleton</td><td>T240</td><td>lyophilized tubulin 99% pure unlabeled from pig brain is purchased (Cytoskeleton) and stored in -80&amp;deg;C until hydrated in PEM80 and used for upto one year.</td></tr><tr><td>UV-Ozone</td><td>Jelight</td><td>Model 342</td><td>For preparing experimental chambers - treating cover glasses</td></tr><tr><td>Stackreg</td><td>Biomedical Imaging Group</td><td>http://bigwww.epfl.ch/thevenaz/stackreg/</td><td>plugin for registering time series data to remove drift</td></tr><tr><td>Turboreg</td><td>Biomedical Imaging Group</td><td>http://bigwww.epfl.ch/thevenaz/turboreg/</td><td>plugin for registering time series data to remove drift</td></tr></tbody></table>

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.

With only a small number of components, tubulin dimers, and microtubule crosslinkers, microtubule tactoids can form (Figure 2A). Although this protocol describes incubation to nucleate and grow microtubules in an incubator, the nucleation and growth can be observed directly under the microscope (which are complete within 30 min) (Figure 2B). The concentration of tubulin is kept constant at 13.6 µm and MAP65-MT binding at 10%.
Figure 4 represents successful data. The tactoids should be visible with both a 561 nm laser in the tubulin channel and 488 nm in the MAP65 channel, which perfectly overlap with each other (Figure 4A). One mystery of the system has been that the width of the tactoids does not seem to vary under a variety of experimental changes, including changing the microtubule lengths, the MAP65 concentration, and the crowding agents (Figure 4B)22,28. The length is much more variable and depends on both the microtubule lengths and the MAP65 concentration (Figure 4B)22,28.
When performing FRAP, it has been observed that the MAP65 signal recovers, but the microtubule signal does not recover (Figure 5). The recovery in FRAP is due to the mobility and movement of the labeled and photobleached objects. In the case of the MAP65, the darkened molecules dissociate and move away from the microtubule and new ones move into the region (Figure 5). The MAP65 binding is in equilibrium, so the rate of binding and unbinding is equal (measured in molecules per second). For the microtubules, no recovery was seen implying that the microtubules are not capable of leaving the tactoid (Figure 5A, Bi, C). Further, no spreading of the darkened region was seen, suggesting that the microtubules are locally immobile and not a fluid within the tactoid shape.
<img alt="Cell division diagram; microtubule spindle formations; metaphase; chromosomes alignment process." class="xfigimg" src="/files/ftp_upload/63952/63952fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Different models of spindle formation. A mitotic spindle is a machine made of microtubules and their associated proteins and enzymes that aligns and separates the chromosomes into the two new daughter cells during cell division. (A) Image of an early Middle Ages drop spindle replica with fine yarn from The Netherlands. This figure has been modified from a Wikimedia image by Peter van der Sluijs30. (B) Three-dimensional reconstruction of microtubules at different stages of wild-type meiosis II. Microtubules are shown in green, and chromosomes are shown in gray. Scale bar = 1 µm. This figure has been modified from Lantzsch et al.31. (C) Microscopy image of the microtubules in a mitotic spindle of a dividing Sf9 cell. The spindle poles and the microtubules of the spindle are labeled with a green fluorescent protein. Scale bar = 5 µm. This figure has been modified from Advani et al.32. (D) Different models of how mitotic and meiotic spindle microtubules are organized. (i) Previously observed for meiotic spindles created from Xenopus egg extracts, the microtubules (green) were deduced to be short and dynamic throughout the spindle. This is similar to a bipolar tactoid organization inside a liquid crystal. (ii) The canonical model for microtubule organization inside a mitotic spindle has two types of microtubules: interpolar or polar microtubules (dark green) that crosslink at the midzone around the chromosomes and kinetochore microtubules (light green) that are bundled and stretched from the pole to the kinetochore to push and pull the chromosomes. In all images, chromosomes are shown in transparent blue, and spindle poles are represented in dark green. (E) Schematics of mesogens (green lines) in a liquid crystal tactoid for (i) bipolar and (ii) homogeneous tactoids. Bipolar tactoids have two poles at the end of the tactoid, and the mesogens re-orient to point to those poles. Homogeneous tactoids have poles at infinity, and the mesogens do not change orientation along the length of the tactoid. <a href="https://www.jove.com/files/ftp_upload/63952/63952fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tactoid formation schematic and micrograph series; tubulin, MAP65, microtubules, time-lapse, 5µm." class="xfigimg" src="/files/ftp_upload/63952/63952fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Microtubule condensation. (A) Microtubules can be bundled and crosslinked by a variety of methods including ionic species, depletion forces caused by crowding agents, and specific microtubule crosslinkers, such as MAP65. (i) Tubulin dimers and MAP65 proteins are mixed to nucleate and grow microtubules. (ii) Microtubules nucleate and grow from the tubulin, and MAP65 immediately binds to microtubules, another MAP65 monomer, or both and causes bundling. (iii) Microtubules in the crosslinked bundles nucleate and grow. (iv) The final configuration is a microtubule tactoid similar to a spindle. (B) Time series of microtubule tactoids nucleating and growing over 105 min. Scale bar = 5 µm. Figure adapted from Edozie et al.22. <a href="https://www.jove.com/files/ftp_upload/63952/63952fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Flow chamber diagram, showing sample flow direction with labeled components for fluid dynamics study." class="xfigimg" src="/files/ftp_upload/63952/63952fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Flow chamber assembly. The flow chamber is made using a glass slide, silanized cover glass, and permanent double-sided tape. The yellow highlighted region is the flow path where the sample is flowed and observed. The volume of the flow chamber is ~20 µL. Epoxy was used to seal the ends of the chamber to keep the sample from evaporating during long-term imaging over several hours. <a href="https://www.jove.com/files/ftp_upload/63952/63952fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tactoid imaging: microtubule, MAP65, merged channels; length-width quantification; Gaussian fit chart." class="xfigimg" src="/files/ftp_upload/63952/63952fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Tactoid images and length and width analysis. (A) Example data of tactoids formed as described and imaged using spinning disc confocal showing (i) microtubule channel imaging rhodamine-labeled tubulin using a 561 nm laser, (ii) GFP-MAP65 channel imaging the GFP using a 488 nm laser, and (iii) merged overlay image from the microtubule channel (magenta) and GFP-MAP65 channel (cyan). Overlap regions are displayed as white and demonstrate that the microtubules and MAP65 colocalize exactly. Scale bar = 10 µm for all images in (A). (B) Quantification of tactoid length and width. (i) Image of a tactoid to be analyzed without labels. Scale bar = 5 µm. (ii) Same image as in (i), where the length (solid line with line caps) and width (dashed line) measurements are denoted. Scale bar = 5 µm. (iii) The width was measured by taking the intensity profile across the tactoid at the perpendicular bisector (dashed line) denoted in (ii). The intensity profile was fit to a Gaussian function to reveal the amplitude and width of the tactoid. <a href="https://www.jove.com/files/ftp_upload/63952/63952fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="FRAP microscopy data and intensity graphs showing microtubule and MAP65 signal recovery analysis." class="xfigimg" src="/files/ftp_upload/63952/63952fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Representative FRAP data and analysis. (A) Microscopy time series data of (i) microtubule tactoid and (ii) GFP-MAP65, and (iii) overlay image of both channels with microtubules in magenta and GFP-MAP65 in cyan that were photobleached at time 63 s and observed for an additional 5 min. (B) Quantified intensity of the (i) microtubule channel in the bleached region (magenta circles) and the background (dark grey circles) and (ii) GFP-MAP65 channel in the bleached region (cyan squares) and the background (dark grey squares). (C) The data were corrected for the background noise and rescaled for the microtubule channel (magenta circles) and the GFP-MAP65 channel (cyan squares). The microtubules do not recover, but the GFP-MAP65 does and can be fit (dark grey line) to a rising exponential decay to find the amplitude and the timescale of recovery. <a href="https://www.jove.com/files/ftp_upload/63952/63952fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Self-Assembly of Microtubule Tactoids at JoVE.com

Self-Assembly of Microtubule Tactoids

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 ...

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Biochemistry

3.7K Views.  Syracuse University. Dit protocol maakt de spontane zelforganisatie van microtubuli tactoïden mogelijk met behulp van een anti-parallelle cross-linker, MAP65. Dit systeem kan ons helpen de organisatie van microtubuli en biologische systemen zoals mitotische spindels te begrijpen. Het belangrijkste voordeel van deze techniek is dat het een minimalistisch systeem is dat spindelachtige vormen voor microtubuli kan recreëren die zeer reproduceerbaar en toegankelijk is voor veel laboratoria.Deze methode is nie...

Video: Self-Assembly of Microtubule Tactoids

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.

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 ...

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 ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

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

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Forming, Confining, and Observing Microtubule-Based Active Nematics

The formation of biopolymer-based active phases has become an important technique for researchers interested in exploring the emerging field of active liquid crystals and their possible roles in cell biology. These novel systems consist of self-driven sub-units that consume energy locally, producing an out-of-equilibrium dynamic fluid. To form the active liquid crystal phase described in this report, purified protein components including biopolymers and molecular motors are combined, and the active nematic phase spontaneously forms in the presence of adenosine triphosphate (ATP). To observe the nematic state, the material must be confined in a suitable geometry for microscopy at a high enough density. This article describes two different methods for the formation of an active nematic phase using microtubules and kinesin motors: assembly of a two-dimensional active layer at an oil and water interface and assembly under an oil layer using an elastomeric well. Techniques to insert the active material into small wells of different shapes are also described.

Presented here are methods for preparing active nematics from microtubules and kinesin motors, including protein preparation and construction and the use of wells for active nematic confinement.

The formation of biopolymer-based active phases has become an important technique for researchers interested in exploring the emerging field of active ...

TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Source: Henty-Ridilla, J.L., <a href="https://app.jove.com/v/64074">Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.</a> J. Vis. Exp. (2022)
This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.

Source: Henty-Ridilla, J.L., Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy. J. ...

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Biological Techniques

Microscopy Techniques

Microtubules

Microtubules are the thickest cytoskeletal filaments with a diameter of 25 nm. In prokaryotic organisms, microtubules are commonly found in locomotory appendages like cilia and flagella. In eukaryotic cells, microtubules form specialized extensions for moving fluid over the surface, like those found in cells lining the intestine.
Microtubules have two structurally similar globular protein subunits: &#945; and &#946; tubulins. In the cytosol, the &#945; and &#946; tubulins form a heterodimer. These &#945;&#946;-heterodimers enter the centrioles, a type of microtubule organizing center (MTOCs), and assemble into a filament with the help of the &#947;-tubulin ring complex. Each microtubule has 13 protofilaments, each with alternating &#945; and &#946; tubulins.
Microtubules are polar structures with plus (+) and minus (&#8722;) ends. The end originating from the MTOCs is the minus end, while the outward-facing end is the plus end. Microtubules are dynamic structures that undergo repeated polymerization and depolymerization. However, these filaments are not flexible; they cannot flex or bend when force is applied, and they will break apart if a deforming force is sufficient.
In microtubules, both &#945;-tubulin and &#946;-tubulin are associated with GTP. The GTP-bound-&#946;-tubulins at the plus end are necessary for microtubule polymerization.&#160; GTP-&#945;-tubulin cannot be hydrolyzed to GDP, however, GTP-&#946;-tubulin may be. The hydrolysis of GTP to GDP breaks the lateral interaction between the protofilaments, allowing tubulin monomers to dissociate.
Microtubules have multiple roles within the eukaryotic cell, and they act as tracks for transporting cargo and vesicles within the cell due to their polar nature. Microtubule-associated motor proteins, such as kinesin and cytoplasmic dyneins, transport cargoes throughout the cell.

Microtubules are the thickest cytoskeletal filaments with a diameter of 25 nm. In prokaryotic organisms, microtubules are commonly found in locomotory ...

Education

JoVE Core

Cell Biology

Unit 1

The Cytoskeleton II: Microtubules and Intermediate Filaments

KEY TERMS AND CONCEPTS

Microtubule Formation

Microtubules are dynamic structures that undergo continuous assembly and disassembly. They originate from specialized multi-protein complexes known as microtubule organizing centers or MTOCs. Within the MTOC, the point of origin of the microtubule is known as the minus end, while the end radiating outward is the plus end. Microtubules serve two primary functions &#8212; the organization of spindle complexes to separate sister chromatids during mitotic or meiotic cell division and the formation of locomotory appendages, like cilia and flagella.
MTOCs are found in both prokaryotic and eukaryotic organisms. However,&#160; some lower eukaryotes, like most fungi, lack organized MTOCs. Instead, they have organized centrosomes consisting of centrioles and the pericentriolar material. In animal cells, the structure and location of MTOCs, vary within different cell types depending on the function of the microtubules.
Microtubule Nucleation
The nucleation of microtubules occurs within the MTOCs, i.e., the centrioles, where different &#947;-tubulin complex proteins interact with &#947;-tubulin subunits to form the &#947;-tubulin-ring complex (&#947;-TRC). Nucleation is initiated when the &#945;-tubulin subunit of the &#945;&#946;-tubulin heterodimer attaches to the &#947;-TRC. Several intrinsic and extrinsic factors influence microtubule nucleation. Intrinsic factors like the &#945;- and &#946;-tubulins isotype incorporated; the concentration of free &#945;&#946;-tubulin heterodimers, the post-translational modifications, and the microtubule-associated proteins (MAPs) affect the microtubule nucleation dynamics. Extrinsic factors like temperature, pH, and microtubule interfering drugs are also responsible for the rate of microtubule polymerization or depolymerization.

Microtubules are dynamic structures that undergo continuous assembly and disassembly. They originate from specialized multi-protein complexes known as ...

Assembly of Cytoskeletal Filaments

Cytoskeletal filaments are polymeric forms of smaller protein subunits. However, individual cytoskeletal filaments may easily disassemble or associate with other similar filaments to form rigid structures. Microfilaments, made of actin monomers, rely on actin-binding proteins to form bundles and create networks of individual actin filaments. Microtubules rely on microtubule-associated proteins (MAPs) to form sturdy cylindrical structures. However, the proteins involved in forming complex rope-like intermediate filament structures are still unknown.&#160;
Assembly of Actin bundles and Networks
Actin bundles can be classified into compact and loosely spaced bundles. Compact bundles consist of closely spaced parallel actin filaments, with their plus ends adjacent to the plasma membranes formed with the help of the actin-binding protein fimbrin, with two adjacent actin-binding domains to hold parallel filaments close together. Loosely spaced bundles known as contractile bundles rely on crosslinking actin-binding proteins such as &#945;-actinin. &#945;-actinin is a homodimeric protein, having a single actin-binding site that binds to two oppositely oriented actin filaments. Actin filaments can also form a network-like structure with the help of larger actin-binding proteins such as filamin. Filamin is a dimeric protein with an actin-binding domain and a dimerization domain at opposite ends of each subunit.&#160; This dimer is in V-shape, with actin-binding domains at the ends of each arm. Therefore, linking actin filament through these proteins form three-dimensional networks.
Assembly of Microtubule Cylinders
Microtubules are cylindrical assemblies of tubulin heterodimers that are several micrometers long and 25 nm wide. They are made up of 13 parallel protofilaments consisting of tubulin dimers arranged head to tail. MAPs such as Tumor Overexpressed Gene or TOG domain-containing microtubule polymerases aid in microtubule protofilaments assembly into cylindrical structures. Some of these TOG domains capture free tubulin, while others anchor the tubulin to the microtubule plus end, favoring the association of protofilaments and promoting microtubule assembly.&#160; &#160;

Cytoskeletal filaments are polymeric forms of smaller protein subunits. However, individual cytoskeletal filaments may easily disassemble or associate ...

Self-Assembly of Microtubule Tactoids

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