KIF1C, an RNA transporting kinesin-3, undergoes liquid-liquid phase separation through its C-terminal disordered domain.

The spatial distribution of mRNA is critical for local control of protein production. Recent studies have identified the kinesin-3 family member KIF1C as an RNA transporter. However, it is not clear how KIF1C interacts with RNA molecules. Here, we show that KIF1C's C-terminal tail domain is an intrinsically disordered region (IDR) containing a prion-like domain (PLD) that is unique compared to the C-terminal tails of other kinesin family members. In cells, KIF1C constructs undergo reversible formation of dynamic puncta that display physical properties of liquid condensates and incorporate RNA molecules in a sequence-selective manner. The IDR is necessary and sufficient for driving liquid-liquid phase separation (LLPS) but the condensate properties can be modulated by adjacent coiled-coil segments. The purified KIF1C IDR domain undergoes LLPS in vitro at near-endogenous nM concentrations in a salt-dependent manner. Deletion of the IDR abolished the ability of KIF1C to undergo LLPS and disrupted the distribution of mRNA cargoes to the cell periphery. Our work thus uncovers an intrinsic correlation between the LLPS activity of KIF1C and its role as an RNA transporter. In addition, as the first kinesin motor reported to undergo LLPS, our work reveals a previously uncharacterized mode of motor-cargo interaction that extends our understanding of the behavior of cytoskeletal motor proteins.

Microtubule detyrosination by VASH1/SVBP is regulated by the conformational state of tubulin in the lattice.

Tubulin, a heterodimer of α- and ß-tubulin, is a GTPase that assembles into microtubule (MT) polymers whose dynamic properties are intimately coupled to nucleotide hydrolysis. In cells, the organization and dynamics of MTs are further tuned by post-translational modifications (PTMs), which control the ability of MT-associated proteins (MAPs) and molecular motors to engage MTs. Detyrosination is a PTM of α-tubulin, wherein its C-terminal tyrosine residue is enzymatically removed by either the vasohibin (VASH) or MT-associated tyrosine carboxypeptidase (MATCAP) peptidases. How these enzymes generate specific patterns of MT detyrosination in cells is not known. Here, we use a novel antibody-based probe to visualize the formation of detyrosinated MTs in real time and employ single-molecule imaging of VASH1 bound to its regulatory partner small-vasohibin binding protein (SVBP) to understand the process of MT detyrosination in vitro and in cells. We demonstrate that the activity, but not binding, of VASH1/SVBP is much greater on mimics of guanosine triphosphate (GTP)-MTs than on guanosine diphosphate (GDP)-MTs. Given emerging data showing that tubulin subunits in GTP-MTs are in expanded conformation relative to tubulin subunits in GDP-MTs, we reasoned that the lattice conformation of MTs is a key factor that gates the activity of VASH1/SVBP. We show that Taxol, a drug known to expand the MT lattice, promotes MT detyrosination and that CAMSAP2 and CAMSAP3 are two MAPs that spatially regulate detyrosination in cells. Collectively, our work shows that VASH1/SVBP detyrosination is regulated by the conformational state of tubulin in the MT lattice and that this is spatially determined in cells by the activity of MAPs.

Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons.

An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin C-terminal tail to interact with effector proteins and are thereby thought to change microtubule dynamics, stability, and organization. The peptidase(s) that produces ΔC2-α-tubulin in a physiological context remains unclear. Here, we take advantage of the observation that ΔC2-α-tubulin accumulates to high levels in cells lacking tubulin tyrosine ligase (TTL) to screen for cytosolic carboxypeptidases (CCPs) that generate ΔC2-α-tubulin. We identify CCP1 as the sole peptidase that produces ΔC2-α-tubulin in TTLΔ HeLa cells. Interestingly, we find that the levels of ΔC2-α-tubulin are only modestly reduced in photoreceptors of ccp1-/- mice, indicating that other peptidases act synergistically with CCP1 to produce ΔC2-α-tubulin in post-mitotic cells. Moreover, the production of ΔC2-α-tubulin appears to be under tight spatial control in the photoreceptor cilium: ΔC2-α-tubulin persists in the connecting cilium of ccp1-/- but is depleted in the distal portion of the photoreceptor. This work establishes the groundwork to pinpoint the function of ΔC2-α-tubulin in proliferating and post-mitotic mammalian cells.

Investigation of mechanisms underlying a light approaching behavior in a house gecko by comparative and learning experiments.

Nocturnal predators of many taxa are known to come to artificial light at night for foraging on clumped food resources. Both innate and acquired light preferences seem to be possible mechanisms of light approaching behavior although empirical tests are lacking in most nocturnal predators. Here, using a Japanese gecko Gekko japonicus, we investigated whether geckos have a light preference and how foraging experiences under the light reinforce light approaching tendency. In a comparative experiment, there was no difference in light approaching behavior between urban and suburban geckos irrespective of their original light habitats. In an associative learning experiment, geckos did not significantly change light approaching behavior even after repeated opportunities to forage crickets near a lamp in the laboratory setting. These results imply that light approaching behavior of Japanese geckos may not be easily reinforced by foraging experiences under the light. Although we often witness geckos coming to artificial light at night, our findings may not suggest their light preference. Geckos may approach the light-up foraging spot based on other cues relating to the artificial light environment.

Two Kinesin-14A Motors Oligomerize to Drive Poleward Microtubule Convergence for Acentrosomal Spindle Morphogenesis in Arabidopsis thaliana.

Plant cells form acentrosomal spindles with microtubules (MTs) converged toward two structurally undefined poles by employing MT minus end-directed Kinesin-14 motors. To date, it is unclear whether the convergent bipolar MT array assumes unified poles in plant spindles, and if so, how such a goal is achieved. Among six classes of Kinesin-14 motors in Arabidopsis thaliana, the Kinesin-14A motors ATK1 (KatA) and ATK5 share the essential function in spindle morphogenesis. To understand how the two functionally redundant Kinesin-14A motors contributed to the spindle assembly, we had ATK1-GFP and ATK5-GFP fusion proteins expressed in their corresponding null mutants and found that they were functionally comparable to their native forms. Although ATK1 was a nuclear protein and ATK5 cytoplasmic prior to nuclear envelop breakdown, at later mitotic stages, the two motors shared similar localization patterns of uniform association with both spindle and phragmoplast MTs. We found that ATK1 and ATK5 were rapidly concentrated toward unified polar foci when cells were under hyperosmotic conditions. Concomitantly, spindle poles became perfectly focused as if there were centrosome-like MT-organizing centers where ATK1 and ATK5 were highly enriched and at which kinetochore fibers pointed. The separation of ATK1/ATK5-highlighted MTs from those of kinetochore fibers suggested that the motors translocated interpolar MTs. Our protein purification and live-cell imaging results showed that ATK1 and ATK5 are associated with each other in vivo. The stress-induced spindle pole convergence was also accompanied by poleward accumulation of the MT nucleator γ-tubulin. These results led to the conclusion that the two Kinesin-14A motors formed oligomeric motor complexes that drove MT translocation toward the spindle pole to establish acentrosomal spindles with convergent poles.

EML2-S constitutes a new class of proteins that recognizes and regulates the dynamics of tyrosinated microtubules.

Tubulin post-translational modifications (PTMs) alter microtubule properties by affecting the binding of microtubule-associated proteins (MAPs). Microtubule detyrosination, which occurs by proteolytic removal of the C-terminal tyrosine from ɑ-tubulin, generates the oldest known tubulin PTM, but we lack comprehensive knowledge of MAPs that are regulated by this PTM. We developed a screening pipeline to identify proteins that discriminate between Y- and ΔY-microtubules and found that echinoderm microtubule-associated protein-like 2 (EML2) preferentially interacts with Y-microtubules. This activity depends on a Y-microtubule interaction motif built from WD40 repeats. We show that EML2 tracks the tips of shortening microtubules, a behavior not previously seen among human MAPs in vivo, and influences dynamics to increase microtubule stability. Our screening pipeline is readily adapted to identify proteins that specifically recognize a wide range of microtubule PTMs.

Tubulin Carboxypeptidase Activity Promotes Focal Gelatin Degradation in Breast Tumor Cells and Induces Apoptosis in Breast Epithelial Cells That Is Overcome by Oncogenic Signaling.

Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

A cytoskeletal function for PBRM1 reading methylated microtubules.

Epigenetic effectors "read" marks "written" on chromatin to regulate function and fidelity of the genome. Here, we show that this coordinated read-write activity of the epigenetic machinery extends to the cytoskeleton, with PBRM1 in the PBAF chromatin remodeling complex reading microtubule methyl marks written by the SETD2 histone methyltransferase. PBRM1 binds SETD2 methyl marks via BAH domains, recruiting PBAF components to the mitotic spindle. This read-write activity was required for normal mitosis: Loss of SETD2 methylation or pathogenic BAH domain mutations disrupt PBRM1 microtubule binding and PBAF recruitment and cause genomic instability. These data reveal PBRM1 functions beyond chromatin remodeling with domains that allow it to integrate chromatin and cytoskeletal activity via its acetyl-binding BD and methyl-binding BAH domains, respectively. Conserved coordinated activity of the epigenetic machinery on the cytoskeleton opens a previously unknown window into how chromatin remodeler defects can drive disease via both epigenetic and cytoskeletal dysfunction.

Prosocial and antisocial choices in a monogamous cichlid with biparental care.

Human society is cooperative and characterized by spontaneous prosociality. Comparative studies on endotherm vertebrates suggest that social interdependence causes the evolution of proactive prosociality. To test the generality of this hypothesis, we modify a prosocial choice task for application to the convict cichlid, Amatitlania nigrofasciata, a monogamous fish with biparental care and a strong pair bond. We also affirm that male subjects learn to favor prosocial choices when their mates are the recipients in a neighboring tank. When the neighboring tank is empty, males choose randomly. Furthermore, in the absence of their mates, males behave prosocially toward a stranger female. However, if the mate of the subjects is also visible in the third tank, or if a male is a potential recipient, then subjects make antisocial choices. To conclude, fish may show both spontaneous prosocial and antisocial behaviors according to their social relationships with conspecifics and the overall social context.

Parthenolide Destabilizes Microtubules by Covalently Modifying Tubulin.

Detyrosination of the α-tubulin C-terminal tail is a post-translational modification (PTM) of microtubules that is key for many biological processes.1 Although detyrosination is the oldest known microtubule PTM,2-7 the carboxypeptidase responsible for this modification, VASH1/2-SVBP, was identified only 3 years ago,8,9 precluding genetic approaches to prevent detyrosination. Studies examining the cellular functions of detyrosination have therefore relied on a natural product, parthenolide, which is widely believed to block detyrosination of α-tubulin in cells, presumably by inhibiting the activity of the relevant carboxypeptidase(s).10 Parthenolide is a sesquiterpene lactone that forms covalent linkages predominantly with exposed thiol groups; e.g., on cysteine residues.11-13 Using mass spectrometry, we show that parthenolide forms adducts on both cysteine and histidine residues on tubulin itself, in vitro and in cells. Parthenolide causes tubulin protein aggregation and prevents the formation of microtubules. In contrast to epoY, an epoxide inhibitor of VASH1/2-SVBP,9 parthenolide does not block VASH1-SVBP activity in vitro. Lastly, we show that epoY is an efficacious inhibitor of microtubule detyrosination in cells, providing an alternative chemical means to block detyrosination. Collectively, our work supports the notion that parthenolide is a promiscuous inhibitor of many cellular processes and suggests that its ability to block detyrosination may be an indirect consequence of reducing the polymerization-competent pool of tubulin in cells.

Affinity purification of tubulin from plant materials.

In the plant cytoskeleton research, mammalian brain tubulin has been widely used to study plant microtubule-interacting proteins in vitro since purification of tubulins from plant sources is generally considered to be challenging and time-consuming. A convenient method for affinity purification of tubulins was devised, which utilized the TOG domains of yeast Stu2 tubulin-binding protein as an affinity ligand (Widlund et al., 2012). We showed that this so-called TOG tubulin affinity chromatography worked efficiently with plant materials, especially actively-dividing cultured cells (Hotta et al., 2016). Plant tubulins purified with the TOG method is highly assembly-competent and thus can be used in various in vitro experiments. Here, we summarize purification strategies of native or tagged plant tubulins as well as an in vitro pull-down assay to monitor their polymerization activity.

Transitive inference in cleaner wrasses (Labroides dimidiatus).

Transitive inference (TI) is the ability to infer unknown relationships from previous information. To test TI in non-human animals, transitive responding has been examined in a TI task where non-adjacent pairs were presented after premise pair training. Some mammals, birds and paper wasps can pass TI tasks. Although previous studies showed that some fish are capable of TI in the social context, it remains unclear whether fish can pass TI task. Here, we conducted a TI task in cleaner wrasses (Labroides dimidiatus), which interact with various client fishes and conspecifics. Because they make decisions based on previous direct and indirect interactions in the context of cleaning interactions, we predicted that the ability of TI is beneficial for cleaner fish. Four tested fish were trained with four pairs of visual stimuli in a 5-term series: A-B+, B-C+, C-D+, and D-E+ (plus and minus denote rewards and non-rewards, respectively). After training, a novel pair, BD (BD test), was presented wherein the fish chose D more frequently than B. In contrast, reinforcement history did not predict the choice D. Our results suggest that cleaner fish passed the TI task, similar to mammals and birds. Although the mechanism underlying transitive responding in cleaner fish remains unclear, this work contributes to understanding cognitive abilities in fish.

Fish focus primarily on the faces of other fish.

"Face" is a special stimulus in humans and, nonhuman primates, and some other social mammals; that is, they perceive the face differently from the other body parts and other stimuli. In these species, the face conveys much information, so individuals examine the face at first sight rather than other body parts. Similar to mammals, the faces of fish also convey much information, but little is known about whether fish pay attention to the face or face-viewing patterns. Here we document the face-viewing patterns of the cichlid fish Neolamprologus brichardi, which can distinguish between conspecifics based on facial colouration. First, we established a method to identify the point at which subject fish inspected. Fish often fixated in direction to their heads toward the object of attention, suggesting that the extended body axis indicated the attention point. Using this attribute, we examined the point of attention of subject fish presented with photographs of conspecifics and heterospecifics. The results revealed that the fish inspected initially and repeatedly at the face and the duration was longer for the face than other body parts.

Polyglutamylation of tubulin's C-terminal tail controls pausing and motility of kinesin-3 family member KIF1A.

The kinesin-3 family member KIF1A plays a critical role in site-specific neuronal cargo delivery during axonal transport. KIF1A cargo is mislocalized in many neurodegenerative diseases, indicating that KIF1A's highly efficient, superprocessive motility along axonal microtubules needs to be tightly regulated. One potential regulatory mechanism may be through posttranslational modifications (PTMs) of axonal microtubules. These PTMs often occur on the C-terminal tails of the microtubule tracks, act as molecular "traffic signals" helping to direct kinesin motor cargo delivery, and include C-terminal tail polyglutamylation important for KIF1A cargo transport. KIF1A initially interacts with microtubule C-terminal tails through its K-loop, a positively charged surface loop of the KIF1A motor domain. However, the role of the K-loop in KIF1A motility and response to perturbations in C-terminal tail polyglutamylation is underexplored. Using single-molecule imaging, we present evidence that KIF1A pauses on different microtubule lattice structures, linking multiple processive segments together and contributing to KIF1A's characteristic superprocessive run length. Furthermore, modifications of the KIF1A K-loop or tubulin C-terminal tail polyglutamylation reduced KIF1A pausing and overall run length. These results suggest a new mechanism to regulate KIF1A motility via pauses mediated by K-loop/polyglutamylated C-terminal tail interactions, providing further insight into KIF1A's role in axonal transport.

If a fish can pass the mark test, what are the implications for consciousness and self-awareness testing in animals?

Abstract: The ability to perceive and recognise a reflected mirror image as self (mirror self-recognition, MSR) is considered a hallmark of cognition across species. Although MSR has been reported in mammals and birds, it is not known to occur in any other major taxon. Potentially limiting our ability to test for MSR in other taxa is that the established assay, the mark test, requires that animals display contingency testing and self-directed behaviour. These behaviours may be difficult for humans to interpret in taxonomically divergent animals, especially those that lack the dexterity (or limbs) required to touch a mark. Here, we show that a fish, the cleaner wrasse Labroides dimidiatus, shows behaviour that may reasonably be interpreted as passing through all phases of the mark test: (i) social reactions towards the reflection, (ii) repeated idiosyncratic behaviours towards the mirror, and (iii) frequent observation of their reflection. When subsequently provided with a coloured tag in a modified mark test, fish attempt to remove the mark by scraping their body in the presence of a mirror but show no response towards transparent marks or to coloured marks in the absence of a mirror. This remarkable finding presents a challenge to our interpretation of the mark test­do we accept that these behavioural responses, which are taken as evidence of self-recognition in other species during the mark test, lead to the conclusion that fish are self-aware? Or do we rather decide that these behavioural patterns have a basis in a cognitive process other than self-recognition and that fish do not pass the mark test? If the former, what does this mean for our understanding of animal intelligence? If the latter, what does this mean for our application and interpretation of the mark test as a metric for animal cognitive abilities? EDITOR'S NOTE: This Short Report received both positive and negative reviews by experts. The Academic Editor has written an accompanying Primer that we are publishing alongside this article (https://doi.org/10.1371/journal.pbio.3000112). The linked Primer presents a complementary expert perspective; it discusses how the current study should be interpreted in the context of evidence for and against self-awareness in a wide range of animals.

Does a cichlid fish process face holistically? Evidence of the face inversion effect.

Faces are the most important body part for differentiating among human individuals by humans. Humans read the face as a whole, rather than looking at its parts, which makes it more difficult to recognise inverted faces than upright. Some other mammals also identify each other based on the upright face and take longer to recognise inverted faces. This effect is called the face inversion effect and is considered as evidence for face-specific perception. This ability has rarely been observed in animals other than mammals, but it was recently reported that some fish species could distinguish among individuals based on the face. For example, the cichlid fish Neolamprologus pulcher rapidly recognises familiar conspecifics by faces rather than other body parts. Here, we examined the face inversion effect in N. pulcher, by showing photographs of conspecific fish faces and objects in both upright and inverted orientations. Subjects gazed at novel faces longer than familiar faces in upright presentation, whereas they did not show such a tendency for inverted faces. Although the object discrimination was difficult, we did not observe the difference between upright and inverted object photographs. Our results indicate that fish exhibits the inversion effect for faces. These findings suggest that N. pulcher may process their conspecifics' face holistically, like humans.

A social cichlid fish failed to pass the mark test.

Since the pioneering work in chimpanzees, mirror self-recognition (MSR), the ability to recognise oneself in a mirror, has been reported in great apes, Asian elephants, dolphins, and some social birds using the mark test, in which animals that possess MSR touch an imperceptible mark on their own bodies only when a mirror is present. However, giant pandas, which are solitary, failed to pass the mark test, suggesting that MSR evolved solely in highly social animals. In contrast to the increasing evidence of MSR in mammals and birds, little is known about MSR in fish. A Tanganyikan cichlid, Neolamprologus pulcher, is a good candidate for study because these fish live in highly social groups and recognise conspecifics about as rapidly as primates. We examined their responses to a mirror image and tested whether N. pulcher could pass the mark test. When the mirror was first exposed, they stayed in front of the mirror and exhibited aggressive behaviour towards the mirror image. These social behaviours suggested that the focal fish perceived the mirror image as an unfamiliar conspecific. The social responses decreased over the following days, as has generally been the case in animals with MSR. After mark injection, we found no increase in scraping behaviour or prolonged observation of the marked side. These results show a lack of contingency checking and mark-directed behaviours, meaning that N. pulcher failed to pass the mark test and did not recognise their self-image in the mirror.

The Mitotic Function of Augmin Is Dependent on Its Microtubule-Associated Protein Subunit EDE1 in Arabidopsis thaliana.

The augmin complex plays an essential role in microtubule (MT)-dependent MT nucleation by recruiting the γ-tubulin complex to MT walls to generate new MTs [1]. The complex contains eight subunits (designated AUG) including AUG8, which is an MT-associated protein (MAP). When this complex is isolated from etiolated seedlings consisting of primarily interphase cells in Arabidopsis thaliana, AUG8 is an integral component [2]. EDE1 (Endosperm DEfective 1) is homologous to AUG8 [3]. Here, we demonstrate that EDE1, but not AUG8, is associated with acentrosomal spindle and phragmoplast MT arrays in patterns indistinguishable from those of the AUG1-7 subunits and the γ-tubulin complex proteins (GCPs) that exhibit biased localization toward MT minus ends. Consistent with this colocalization, EDE1 directly interacts with AUG6 in vivo. Moreover, a partial loss-of-function mutation, ede1-1, compromises the localization of augmin and γ-tubulin on the spindle and phragmoplast MT arrays and leads to serious distortions in spindle MT remodeling during mitosis. However, mitosis continues even when kinetochore fibers are not obviously discernable, and cytokinesis takes place following the formation of elongated bipolar phragmoplast MT arrays in the mutant. Hence, we conclude that the mitotic function of augmin is dependent on its MAP subunit EDE1, which cannot be replaced by AUG8, and that the cell-cycle-dependent function of augmin can be differentially regulated by employing distinct MAP subunits. Our results also illustrate that plant cells can respond flexibly to serious challenges of compromised MT-dependent MT nucleation to complete mitosis and cytokinesis.

Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization.

Plant cortical microtubules align perpendicular to the growth axis to determine the direction of cell growth. However, it remains unclear how plant cells form well-organized cortical microtubule arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell expansion. Quantitative analysis of hypocotyl cell growth in the nek6-1 mutant demonstrated that NEK6 suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl. Loss of NEK6 function led to excessive microtubule waving and distortion, implying that NEK6 suppresses the aberrant cortical microtubules. Live cell imaging showed that NEK6 localizes to the microtubule lattice and to the shrinking plus and minus ends of microtubules. In agreement with this observation, the induced overexpression of NEK6 reduced and disorganized cortical microtubules and suppressed cell elongation. Furthermore, we identified five phosphorylation sites in ß-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant ß-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of ß-tubulin and the resulting destabilization of cortical microtubules.

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco.

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.