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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;l aliquots in -20&#176;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&#176;C</td>
			<td>Fisherbrand</td>
			<td>13986148</td>
			<td>For storing reagents</td>
		</tr>
		<tr>
			<td>Freezer -80&#176;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 &#956;l aliquots in -20&#176;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&#176;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&#176;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 &#956;M solution. This working solution is stored at 4&#176;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&#176;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&#176;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&#176;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&#176;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&#176;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 &#181;m 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="Figure 1" class="xfigimg" src="/files/ftp_upload/63952/63952fig01.jpg" /> 
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 &#181;m. 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 &#181;m. 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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63952/63952fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63952/63952fig02.jpg" /> 
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)&#160;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)&#160;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 &#181;m. Figure adapted from Edozie et al.22. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63952/63952fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63952/63952fig03.jpg" /> 
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 &#181;L. 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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63952/63952fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63952/63952fig4.jpg" /> 
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 &#181;m for all images in (A). (B) Quantification of tactoid length and width.&#160;(i) Image of a tactoid to be analyzed without labels. Scale bar = 5 &#181;m. (ii) Same image as in (i), where the length (solid line with line caps) and width (dashed line) measurements are denoted. Scale bar = 5 &#181;m. (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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63952/63952fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63952/63952fig05.jpg" /> 
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)&#160;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://jove.unicartagenaproxy.elogim.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 ...

Research

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Biochemistry

3.6K Views.  Syracuse University. 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.

Video: Self-Assembly of Microtubule Tactoids

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

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 ...

Self-Assembly of Microtubule Tactoids

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Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Practical Aspects of Sample Preparation and Setup of ¹H R_1ρ Relaxation Dispersion Experiments of RNA

Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

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