Relating nucleotide‐dependent conformational changes in free tubulin dimers to tubulin assembly

The complex dynamic behavior of microtubules (MTs) is believed to be primarily due to the αβ‐tubulin dimer architecture and its intrinsic GTPase activity. Hence, a detailed knowledge of the conformational variations of isolated α‐GTP‐β‐GTP‐ and α‐GTP‐β‐GDP‐tubulin dimers in solution and their implications to interdimer interactions and stability is directly relevant to understand the MT dynamics. An attempt has been made here by combining molecular dynamics (MD) simulations and protein–protein docking studies that unravels key structural features of tubulin dimer in different nucleotide states and correlates their association to tubulin assembly. Results from simulations suggest that tubulin dimers and oligomers attain curved conformations in both GTP and GDP states. Results also indicate that the tubulin C‐terminal domain and the nucleotide state are closely linked. Protein–protein docking in combination with MD simulations suggest that the GTP‐tubulin dimers engage in relatively stronger interdimer interactions even though the interdimer interfaces are bent in both GTP and GDP tubulin complexes, providing valuable insights on in vitro finding that GTP‐tubulin is a better assembly candidate than GDP‐tubulin during the MT nucleation and elongation processes. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 282–291, 2013.     

Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP

The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends.     

Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs.     

Microtubule-associated proteins promote microtubule generation in the absence of γ-tubulin in human colon cancer cells

The γ-tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the γ-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerization/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in γ-tubulin–independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organized noncentriolar MT organizing centers (ncMTOCs) in the absence of γ-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that these proteins might promote MT nucleation in the absence of γ-tubulin. In contrast, depletion of ch-TOG or CAMSAPs did not affect the timing of ncMTOC appearance. CLASP1 also accelerates γ-tubulin–independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the γ-tubulin template.     

Effect of tubulin self-association on GTP hydrolysis and nucleotide exchange reactions

We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. Both reactions are relevant for microtubule (MT) dynamics. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied by determining the concentrations of tubulin-free and tubulin-bound GTP and GDP. We observed no GTP hydrolysis below the critical conditions for MT assembly (either below the critical tubulin concentration and/or at low temperature), despite the assembly of tubulin 1D curved oligomers and single-rings, showing that their assembly did not involve GTP hydrolysis. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Cryo-TEM images showed that GTP-tubulin 1D oligomers were curved also at 36 °C. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to determine the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites. This conclusion may have implications on the dynamics at the tip of the MT plus end.     

CLAMP, a novel microtubule-associated protein with EB-type calponin homology

Microtubules (MTs) are polymers of alpha and beta tubulin dimers that mediate many cellular functions, including the establishment and maintenance of cell shape. The dynamic properties of MTs may be influenced by tubulin isotype, posttranslational modifications of tubulin, and interaction with microtubule-associated proteins (MAPs). End-binding (EB) family proteins affect MT dynamics by stabilizing MTs, and are the only MAPs reported that bind MTs via a calponin-homology (CH) domain (J Biol Chem 278 (2003) 49721-49731; J Cell Biol 149 (2000) 761-766). Here, we describe a novel 27 kDa protein identified from an inner ear organ of Corti library. Structural homology modeling demonstrates a CH domain in this protein similar to EB proteins. Northern and Western blottings confirmed expression of this gene in other tissues, including brain, lung, and testis. In the organ of Corti, this protein localized throughout distinctively large and well-ordered MT bundles that support the elongated body of mechanically stiff pillar cells of the auditory sensory epithelium. When ectopically expressed in Cos-7 cells, this protein localized along cytoplasmic MTs, promoted MT bundling, and efficiently stabilized MTs against depolymerization in response to high concentration of nocodazole and cold temperature. We propose that this protein, designated CLAMP, is a novel MAP and represents a new member of the CH domain protein family.     

Centaurin-α2 Interacts with β-Tubulin and Stabilizes Microtubules

Centaurin-α₂ is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α₂ can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α₂ interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α₂ overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α₂ mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α₂ remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α₂ and tubulin confirmed that Centaurin-α₂ promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α₂ overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α₂ role on MT stabilization. Centaurin-α₂ interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α₂ as a new microtubule-associated protein (MAP) increasing MT stability.     

Microtubule nucleation from stable tubulin oligomers

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.     

Reduction in Microtubule Dynamics In Vitro by Brain Microtubule-Associated Proteins and by a Microtubule-Associated Protein-2 Second Repeated Sequence Analogue

Abstract: Microtubule-associated protein (MAP) binding to assembled microtubules (MTs) can be reduced by the addition of polyglutamate without significant MT depolymerization or interference with MT elongation reactions. Ensuing polymer length redistribution in MAP-depleted MTs occurs on a time scale characteristic of that observed with MAP-free MTs. The redistribution phase occurs even in the absence of mechanical shearing and without appreciable effects from end-to-end annealing, as indicated by the time course of incremental changes in polymer length and MT number concentration. We also observed higher rates of MT length redistribution when the [MAP]/[tubulin] ratio was decreased. Together, these results demonstrate that MT length redistribution rates are greatly influenced by MAP content, and the data are compatible with the dynamic instability model. We also found that a peptide analogue corresponding to the second repeated sequence in the MT-binding region of MAP-2 can also markedly retard MT length redistribution kinetics, a finding that accords with the ability of this peptide to promote tubulin polymerization in the absence of MAPs and to displace MAP-2 from MTs. These results provide further evidence that MAPs can modulate MT assembly/disassembly dynamics and that peptide analogues can mimic the action of intact MAPs without the need for three contiguous repeated sequences in the MT-binding region.     

The γ -Tubulin Complex Protein GCP4 Is Required for Organizing Functional Microtubule Arrays in Arabidopsis thaliana

Microtubule (MT) nucleation and organization depend on the evolutionarily conserved protein γ -tubulin, which forms a complex with GCP2-GCP6 (GCP for γ -Tubulin Complex Protein). To date, it is still unclear how GCP4-GCP6 (the non-core GCPs) may be involved in acentrosomal MT nucleation in plant cells. We found that GCP4 was associated with γ -tubulin in vivo in Arabidopsis thaliana. When GCP4 expression was repressed by an artificial microRNA, transgenic plants exhibited phenotypes of dwarfism and reduced organ size. In mitotic cells, it was observed that the γ -tubulin signal associated with the mitotic spindle, and the phragmoplast was depleted when GCP4 was downregulated. Consequently, MTs failed to converge at unified spindle poles, and the bipolar phragmoplast MT array frequently had discrete bundles with extended minus ends, resulting in failed cytokinesis as reflected by cell wall stubs in leaf epidermal cells. In addition, cortical MTs in swollen guard cells and pavement cells of the leaf epidermis became hyperparallel and bundled, which was likely caused by frequent MT nucleation with shallow angles on the wall of extant MTs. Therefore, our results support the notion that GCP4 is an indispensable component for the function of γ -tubulin in MT nucleation and organization in plant cells.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.     

Microtubule Simulations Provide Insight into the Molecular Mechanism Underlying Dynamic Instability

The dynamic instability of microtubules (MTs), which refers to their ability to switch between polymerization and depolymerization states, is crucial for their function. It has been proposed that the growing MT ends are protected by a “GTP cap” that consists of GTP-bound tubulin dimers. When the speed of GTP hydrolysis is faster than dimer recruitment, the loss of this GTP cap will lead the MT to undergo rapid disassembly. However, the underlying atomistic mechanistic details of the dynamic instability remains unclear. In this study, we have performed long-time atomistic molecular dynamics simulations (1 μs for each system) for MT patches as well as a short segment of a closed MT in both GTP- and GDP-bound states. Our results confirmed that MTs in the GDP state generally have weaker lateral interactions between neighboring protofilaments (PFs) and less cooperative outward bending conformational change, where the difference between bending angles of neighboring PFs tends to be larger compared with GTP ones. As a result, when the GDP state tubulin dimer is exposed at the growing MT end, these factors will be more likely to cause the MT to undergo rapid disassembly. We also compared simulation results between the special MT seam region and the remaining material and found that the lateral interactions between MT PFs at the seam region were comparatively much weaker. This finding is consistent with the experimental suggestion that the seam region tends to separate during the disassembly process of an MT.     

Intrinsic bending of microtubule protofilaments

The complex polymerization dynamics of the microtubule (MT) plus end are closely linked to the hydrolysis of the GTP nucleotide bound to the β-tubulin. The destabilization is thought to be associated with the conformational change of the tubulin dimers from the straight conformation in the MT lattice to a curved conformation. It remains under debate whether this transformation is directly related to the nucleotide state, or a consequence of the longitudinal or lateral contacts in the MT lattice. Here, we present large-scale atomistic simulations of short tubulin protofilaments with both nucleotide states, starting from both extreme conformations. Our simulations indicate that both interdimer and intradimer contacts in both GDP and GTP-bound tubulin dimers and protofilaments in solution bend. There are no observable differences between the mesoscopic properties of the contacts in GTP and GDP-bound tubulin or the intradime and interdimer interfaces.     

Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly

Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder''s axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.     

Straightening up is required to nucleate new microtubules

In this issue, Ayukawa, Iwata, Imai, and colleagues (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202007033) use rapid temporal and high-spatial-resolution electron microscopy imaging to examine the earliest stages of new microtubule nucleation. They discover that straightening of curved tubulin oligomers increases the efficiency of microtubule nucleation.

Microtubules are long cytoskeletal filaments that provide structure to cells, allow for transport within the cells, and participate in cell division by acting together with molecular motors to build a mitotic spindle. While microtubules act as the stiff “bones” of the cell, they also have a unique and important ability to rapidly restructure their length and organization in response to cellular cues. Therefore, large arrays of microtubules, such as in a mitotic spindle, can rapidly depolymerize and disappear as needed. However, in order to rebuild these microtubule networks, the nucleation of new microtubules is required. This nucleation of new microtubules, whether from existing templates such as centrosomes or spindle poles, or via the de novo organization of the tubulin subunits that make up microtubules, remains a poorly understood process.In this issue, Ayukawa, Iwata, Imai, and colleagues used rapid temporal and high-spatial-resolution imaging to study the earliest stages of microtubule nucleation (1). First, the authors purified αβ-tubulin heterodimers with a Y222F mutation in the β-tubulin subunit. This Y222F β-tubulin mutation increased the rate of microtubule assembly and, importantly, greatly accelerated the nucleation rate of new microtubules. Thus, a comparison between wild-type and mutant tubulin allowed the authors to dissect differences in the early nucleation process that could explain the increased nucleation rate for the mutant tubulin. Importantly, a “rapid flush method” was used to capture high-resolution transmission electron microscopy images of tubulin subunits very early in the nucleation process. The rapid flush method revealed “oligomers” of tubulin subunits: chains of tubulin subunits linked together along their long axis.The authors reasoned that these oligomers are likely on-pathway intermediates that are crucial for new microtubule nucleation. Examination of the oligomers revealed differences in curvature between wild-type and mutant tubulin: the mutant tubulin that nucleated more readily had oligomers that were straighter and less curled than the wild-type tubulin. Further, a comparison of GTP tubulin and GDP tubulin early nucleation intermediates revealed that, for both wild-type and mutant tubulin, the GDP tubulin oligomers, which did not nucleate efficiently, were more curved than the GTP tubulin oligomers that nucleated more efficiently (Fig. 1 A, left, red versus blue). Importantly, the fraction of nearly straight oligomers in each sample directly corresponded to the respective nucleation rate for each tubulin type. Thus, the degree of curvature of the oligomers predicted the overall nucleation rate, such that an increase in the fraction of straight oligomers was directly correlated to an increase in the microtubule nucleation rate (Fig. 1 A).Open in a separate windowFigure 1.Long, straight oligomers promote microtubule nucleation. (A) The nucleation of new microtubules is limited by the availability of critical-length, straight GTP tubulin (blue) oligomers. GDP tubulin oligomers (red) are more curved, with reduced nucleation efficiency. (B) Straight GTP tubulin protofilaments (blue) that are attached to templates (gray) could also be required to facilitate the nucleation of new microtubules from templates such as in centrosomes. Curved GDP tubulin protofilaments (red) attached to templates (gray) would not efficiently facilitate nucleation of new microtubules.The authors then examined the role of oligomer length in the nucleation rate, to determine whether there was a critical minimum oligomer length that could predict efficient microtubule nucleation. They fit their bulk microtubule growth curves (turbidity) to a standard nucleation-and-growth model (2) to estimate the minimum size of the oligomers that were likely to grow into microtubules, i.e., the critical length. For wild-type tubulin, ∼4 tubulin dimers were required in order for oligomers to grow into microtubules. However, this critical length was common, and indeed prevalent, within the early nucleation mixtures. Therefore, it seems likely that oligomer curvature, rather than length, is the limiting factor that slows the microtubule nucleation rate (Fig. 1 A).Why would oligomer curvature limit microtubule nucleation rate? While oligomers of sufficient length were readily observed in early nucleation mixtures, lateral association of new oligomers with existing oligomers was rare. Further, when multiple oligomers had indeed associated laterally to form a new, multi-protofilament assembly, the length of the longest strand greatly exceeded the maximum size of the single-stranded oligomers. Thus, it is likely that straight oligomers facilitate the lateral association of new tubulin subunits or oligomers along their length, stabilizing the nascent microtubules and allowing for their stable growth as a multistranded filament.While this work sheds light on the nucleation of new microtubules using purified tubulin, the described results provide interesting insights into potential nucleation mechanisms inside of cells. In cells, microtubules predominately grow from templates, such as centrosomes, and various microtubule-associated proteins may also influence the nucleation process. A recent study of microtubule nucleation from templates found that there was a significant time lag between the arrival of new tubulin subunits to a template and the growth of a new microtubule from the template (3). That study concluded that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent microtubule. These results are consistent with the conclusion from Ayukawa, Iwata, Imai, and colleagues that GDP tubulin oligomers are curved and therefore unable to efficiently make lateral associations with other oligomers to stabilize the new microtubule and allow for growth (Fig. 1 B; 1). However, recent electron microscopy studies have also revealed that the GTP tubulin–containing ends of growing microtubules show extended protofilaments with a gentle curvature (4, 5, 6, 7, 8). Thus, one additional barrier to the nucleation of new microtubules on templates may be the straightening of GTP tubulin oligomers. Here, the straightening of template-attached, gently curved GTP tubulin oligomers would then allow for lateral binding of new tubulin dimers and oligomers (Fig. 1 B). The transient straightening of gently curved GTP tubulin oligomers could potentially be accomplished via thermal forces and the resulting protofilament curvature fluctuations. However, this straightening process could also be facilitated by microtubule-associated proteins that promote nucleation, such as TPX2 and XMAP215 (9, 10). In support of this idea, Ayukawa, Iwata, Imai, and colleagues found that the distribution of oligomer curvatures appeared similar to what has been reported for the protofilaments at the growing ends of microtubules (5).In light of these results, interesting future work could explore whether microtubule-associated proteins act to regulate the curvature of the oligomers involved in microtubule nucleation. While many aspects of the mechanisms of microtubule nucleation remain unknown, this new work sheds light on the very earliest stages of microtubule nucleation, which may have wide-ranging implications in future studies of microtubule nucleation and growth in cells.     

Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome

Background

Microtubules (MTs) are assembled by heterodimers of α- and β-tubulins, which provide tracks for directional transport and frameworks for the spindle apparatus and the phragmoplast. MT nucleation and dynamics are regulated by components such as the γ-tubulin complex which are conserved among eukaryotes, and other components which are unique to plants. Following remarkable progress made in the model plant Arabidopsis thaliana toward revealing key components regulating MT activities, the completed rice (Oryza sativa) genome has prompted a survey of the MT cytoskeleton in this important crop as a model for monocots.

Scope

The rice genome contains three α-tubulin genes, eight β-tubulin genes and a single γ-tubulin gene. A functional γ-tubulin ring complex is expected to form in rice as genes encoding all components of the complex are present. Among proteins that interact with MTs, compared with A. thaliana, rice has more genes encoding some members such as the MAP65/Ase1p/PRC1 family, but fewer for the motor kinesins, the end-binding protein EB1 and the mitotic kinase Aurora. Although most known MT-interacting factors have apparent orthologues in rice, no orthologues of arabidopsis RIC1 and MAP18 have been identified in rice. Among all proteins surveyed here, only a few have had their functions characterized by genetic means in rice. Elucidating functions of proteins of the rice MT cytoskeleton, aided by recent technical advances made in this model monocot, will greatly advance our knowledge of how monocots employ their MTs to regulate their growth and form.Key words: Cytoskeleton, kinesins, microtubules (MTs), microtubule-associated proteins (MAPs), motors, rice, Oryza sativa     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Acetylation/deacetylation and microtubule associated proteins influence flagellar axonemal stability and sperm motility

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.     

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4°C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60–68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.