Tubulin Complex Research Articles

Transport and diffusion of Tau protein in neurons.

In highly polarized and elongated cells such as neurons, Tau protein must enter and move down the axon to fulfill its biological task of stabilizing axonal microtubules. Therefore, cellular systems for distributing Tau molecules are needed. This review discusses different mechanisms that have been proposed to contribute to the dispersion of Tau molecules in neurons. They include (1) directed transport along microtubules as cargo of tubulin complexes and/or motor proteins, (2) diffusion, either through the cytosolic space or along microtubules, and (3) mRNA-based mechanisms such as transport of Tau mRNA into axons and local translation. Diffusion along the microtubule lattice or through the cytosol appear to be the major mechanisms for axonal distribution of Tau protein in the short-to-intermediate range over distances of up to a millimetre. The high diffusion coefficients ensure that Tau can distribute evenly throughout the axonal volume as well as along microtubules. Motor protein-dependent transport of Tau dominates over longer distances and time scales. At low near-physiological levels, Tau is co-transported along with short microtubules from cell bodies into axons by cytoplasmic dynein and kinesin family members at rates of slow axonal transport.

Structural Basis of Microtubule Stabilization by Laulimalide and Peloruside A

Laulimalide and peloruside A are microtubule-stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X-ray crystallography it is shown that laulimalide and peloruside A bind to a unique non-taxane site on β-tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane-site M-loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M-loop of β-tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.

Structural Basis of Microtubule Stabilization by Laulimalide and Peloruside A

AbstractLaulimalide and peloruside A are microtubule‐stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X‐ray crystallography it is shown that laulimalide and peloruside A bind to a unique non‐taxane site on β‐tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane‐site M‐loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M‐loop of β‐tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.

Structure of a kinesin–tubulin complex and implications for kinesin motility

The typical function of kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a kinesin's load along a microtubule, we determined the X-ray structure of human kinesin-1 bound to αβ-tubulin. The structure defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the kinesin motor domain from microtubules. It also reveals the structural linkages that connect the ATP nucleotide to the kinesin neck linker, a 15-amino acid segment C terminal to the catalytic core of the motor domain, to result in the power stroke. ATP binding to the microtubule-bound kinesin favors neck-linker docking. This biases the attachment of kinesin's second head in the direction of the movement, thus initiating each of the steps taken.

Structural Model for Tubulin Recognition and Deformation by Kinesin-13 Microtubule Depolymerases

To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.

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.

Development of robust QSAR model using rapid overlay of crystal structures (ROCS) based alignment: a test case of Tubulin inhibitors

Conformational alignment is the most important step in 3D-QSAR methodology. There are various methods available to create an alignment hypothesis for the compounds being studied in 3D-QSAR. Most famous alignment methods include: alignment on a suitable template conformation, field fit alignment, database alignment, pharmacophore-based alignment, atom fit, and docking based alignment. All of the above-mentioned methods were explored previously to sample the conformational space in 3D-QSAR. Current study is dealing with the situation where most of the alignment methods were failed to produce a statistically robust 3D-QSAR model. Therefore, a relatively new alignment scheme was employed using ROCS (Rapid Overlay of Chemical Structures) method from OpenEyes. In the present study, Combretastatin A-4 based Tubulin inhibitors were used as case study. Co-crystal ligand conformation (i.e., Colchicine) in the Tubulin complex structure (PDB ID: 1SA0) was utilized as a template molecule. To date, very few applications of ROCS-based alignments in 3D-QSAR are reported in literature. Therefore, current study will serve as an addition to the existing status of using ROCS in 3D-QSAR. Genetic algorithm was utilized to build final 3D-QSAR model. Correlation obtained from final model suggesting that robust 3D-QSAR model was constructed.

TRPV1–tubulin complex: involvement of membrane tubulin in the regulation of chemotherapy‐induced peripheral neuropathy

Existence of microtubule cytoskeleton at the membrane and submembranous regions, referred as 'membrane tubulin' has remained controversial for a long time. Since we reported physical and functional interaction of Transient Receptor Potential Vanilloid Sub Type 1 (TRPV1) with microtubules and linked the importance of TRPV1-tubulin complex in the context of chemotherapy-induced peripheral neuropathy, a few more reports have characterized this interaction in in vitro and in in vivo condition. However, the cross-talk between TRPs with microtubule cytoskeleton, and the complex feedback regulations are not well understood. Sequence analysis suggests that other than TRPV1, few TRPs can potentially interact with microtubules. The microtubule interaction with TRPs has evolutionary origin and has a functional significance. Biochemical evidence, Fluorescence Resonance Energy Transfer analysis along with correlation spectroscopy and fluorescence anisotropy measurements have confirmed that TRPV1 interacts with microtubules in live cell and this interaction has regulatory roles. Apart from the transport of TRPs and maintaining the cellular structure, microtubules regulate signaling and functionality of TRPs at the single channel level. Thus, TRPV1-tubulin interaction sets a stage where concept and parameters of 'membrane tubulin' can be tested in more details. In this review, I critically analyze the advancements made in biochemical, pharmacological, behavioral as well as cell-biological observations and summarize the limitations that need to be overcome in the future.

Tubulin-based Structure-affinity Relationships for Antimitotic Vinca Alkaloids

The Vinca alkaloids are a group of widely used anticancer drugs, originally extracted from the Madagascar periwinkle, that disrupt microtubule dynamics in mammalian cells by interfering with proper assembly of α,β-tubulin heterodimers. They favor curved tubulin assemblies that destabilize microtubules and induce formation of spiral aggregates. Their binding energy profiles have been characterized by means of sedimentation velocity assays and the binding site of vinblastine at the interface between two tubulin dimers (α1β1 � α2β2) has been ascertained by X-ray crystallographic studies on a complex of tubulin with the stathmin-like domain of protein RB3, albeit at relatively low resolution. Here we use molecular modeling and simulation techniques to build, refine and perform a comparative analysis of the three-dimensional complexes of vinblastine, vincristine, vinorelbine and vinflunine with a β1α2-tubulin interface in explicit water to rationalize the binding affinity differences in structural and energetic terms. Our results shed some more light into the binding determinants and the structure-activity relationships of these clinically useful agents.

Binding of neuronal α-synuclein to β-III tubulin and accumulation in a model of multiple system atrophy

Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. We generated a transgenic (Tg) mouse model in which human α-syn was overexpressed in oligodendrocytes. Our previous studies have revealed that oligodendrocytic α-syn inclusions induced neuronal α-syn accumulation, thereby resulting in progressive neuronal degeneration in mice. We also demonstrated that an insoluble complex of α-syn and β-III tubulin in microtubules progressively accumulated in neurons, thereby leading to neuronal degeneration. In the present study, we demonstrated that neuronal accumulation of the insoluble complex was derived from binding of α-syn to β-III tubulin and not from α-syn self-aggregation. Thus, interaction between α-syn and β-III tubulin plays an important role in neuronal α-syn accumulation in an MSA mouse model.

Mechanistic Logic Underlying the Axonal Transport of Cytosolic Proteins

Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivatable GFP (PAGFP) move with a slow motor-dependent anterograde bias distinct from both vesicular trafficking and diffusion of untagged PAGFP. The overall bias is likely generated by an intricate particle kinetics involving transient assembly and short-range vectorial spurts. In vivo biochemical studies reveal that cytosolic proteins are organized into higher order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supramolecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multiprotein complexes that are directly/indirectly conveyed by motors.

Microtubule Reorganization during Mitosis and Cytokinesis: Lessons Learned from Developing Microgametophytes in Arabidopsis Thaliana

In angiosperms, mitosis and cytokinesis take place in the absence of structurally defined microtubule-organizing centers and the underlying mechanisms are largely unknown. In the spindle and phragmoplast, microtubule reorganization depends on microtubule-interacting factors like the γ-tubulin complex. Because of their critical functions in cell division, loss-of-function mutations in the corresponding genes are often homozygous or sporophytic lethal. However, a number of mutations like gem1, gcp2, and nedd1 can be maintained in heterozygous mutants in Arabidopsis thaliana. When mutant microspores produced by a heterozygous parent undergo pollen mitosis I, they are amenable for phenotypic characterization by fluorescence microscopy. The results would allow us to pinpoint at specific functions of particular proteins in microtubule reorganization that are characteristic to specific stages of mitosis and cytokinesis. Conclusions made in the developing microgametophytes can be extrapolated to somatic cells regarding mechanisms that regulate nuclear migration, spindle pole formation, phragmoplast assembly, and cell division plane determination.

C6-ceramide synergistically potentiates the anti-tumor effects of histone deacetylase inhibitors via AKT dephosphorylation and α-tubulin hyperacetylation both in vitro and in vivo

Histone deacetylase inhibitors (HDACIs) have shown promising anti-tumor effects for a variety of malignancies, however, many tumors are reportedly resistant to them. In this study, we made a novel discovery that co-administration of HDACIs (Trichostatin A (TSA) and others) and exogenous cell-permeable short-chain ceramide (C6) results in striking increase in cancer cell death and apoptosis in multiple cancer cells. These events are associated with perturbations in diverse cell signaling pathways, including inactivation of Akt/mTOR and increase in α-tubulin acetylation (both in vivo and in vitro). TSA interacts in a highly synergistic manner with C6-ceramide to disrupt HDAC6/protein phosphatase 1 (PP1)/tubulin complex, to induce α-tubulin hyperacetylation, and to release and activate PP1, which then leads to AKT dephosphorylation and eventually causes cancer cell death. Interestingly, TSA itself results in short-term ceramide accumulation, which as a result of metabolic (glycosylation) removal, does not result in evident increase of cancer cell death. However, adding C6-ceramide led to a very pronounced increase in ceramide level and marked increase in cell death. Importantly, the effective synergistic anti-tumor activity of TSA plus C6-ceramide is also seen in in vivo mice xenograft pancreatic and ovarian cancer models, indicating that this regimen (HDACI plus C6-ceramide) may represent a more effective form of therapy against pancreatic and ovarian carcinoma.

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

Microtubule (MT) nucleation and organization depend on the evolutionarily conserved protein gamma -tubulin, which forms a complex with GCP2-GCP6 (GCP for gamma -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 gamma -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 gamma -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 gamma -tubulin in MT nucleation and organization in plant cells.

Auditory development in progressive motor neuronopathy mouse mutants

The present study was performed to elucidate the hearing development in the progressive motor neuronopathy ( pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance.

Complexes of tubulin with inhibitors that bind to the colchicine domain

Complexes of tubulin with inhibitors that bind to the colchicine domain

Macromolecular Interaction of Halichondrin B Analogues Eribulin (E7389) and ER-076349 with Tubulin by Analytical Ultracentrifugation

Halichondrin B is an antimitotic drug that inhibits microtubule assembly. To understand the molecular details of its interaction with tubulin, we investigated the binding of two halichondrin B analogues, eribulin (previously, ER-086526, E7389) and ER-076349, to tubulin by quantitative analytical ultracentrifugation. Eribulin is currently undergoing phase III clinical trials for cancer; ER-076349 is a closely related analogue with C.35 hydroxyl instead of C.35 primary amine [Towle, M. J., et al. (2001) Cancer Res. 61, 1013]. Below the critical concentration for microtubule assembly and in the presence of GDP, tubulin undergoes weak self-association into short curved oligomers. Eribulin inhibits this oligomer formation 4-6-fold, while ER-076349 slightly stimulates oligomer formation by 2-fold. This is in contrast to vinblastine which strongly stimulates large spiral polymers by 1000-fold under these same conditions. Vinblastine-induced spiral formation is strongly inhibited by both eribulin and ER-076349. Colchicine binding to the intradimer interface has no significant effect on small oligomer formation or the inhibitory activity of eribulin on this process. These results suggest that halichondrin B analogues bind to the interdimer interface or to the beta-subunit alone, disrupt polymer stability, and compete with vinblastine-induced spiral formation. Stathmin is known to form a tight 1:2 complex with tubulin. Eribulin strongly inhibits formation of the 1:2 stathmin-tubulin complex (>3.3 kcal/mol), while ER-076349 weakens formation of the 1:2 complex by approximately 1.9 kcal/mol. These results suggest that eribulin is a global inhibitor of tubulin polymer formation, disrupting tubulin-tubulin contacts at the interdimer interface. ER-076349 also perturbs tubulin-tubulin contacts, but in a more polymer specific manner, reflecting adaptability of the interdimer interface to drug and polymer polymorphism. These results suggest halichondrin B analogues exhibit unique tubulin-based activities that may underlie the clinical utility of these compounds.

Interaction forces and interface properties of KIF1A kinesin- αβ tubulin complex assessed by molecular dynamics

Kinesin is a microtubule-based motor protein that generates motion involved in intracellular trafficking and cell division. Even if the force-generating and enzymatic properties of kinesin were extensively studied, the molecular basis of its interaction with the microtubule is still not well understood. The aim of the present study is to provide a detailed description, in terms of conformational changes and interaction properties, of the kinesin– αβ tubulin complex during a cycle of ATP hydrolysis. Four different nucleotide-dependent conformations (nucleotide-free, ATP, ADP·Pi and ADP) of the kinesin– αβ tubulin were constructed and investigated by performing molecular dynamics simulations. Computational results show that small conformational changes, in the order of few Angstrom, occurring in the kinesin structure reflect on its affinity for the filament substrate. Indeed the rotation of the α4 helix due to the transition from the bound (ADP·Pi) to the unbound (ADP) state, when the Pi is released from the complex, coupled with the modification occurred in the loop L9 of switch I domain are associated to a marked decrease (approximately 45%) of the maximum interaction force between the kinesin motor and the tubulin dimer.

A comparative molecular field and comparative molecular similarity indices analyses (CoMFA and CoMSIA) of N-phenyl- N′-(2-chloroethyl)ureas targeting the colchicine-binding site as anticancer agents

To decipher the mechanism underlying the covalent binding of N-phenyl- N′-(2-chloroethyl)ureas (CEU) to the colchicine-binding site on β II-tubulin and to design new and selective antimitotic drugs, we developed 3D quantitative structure–activity relationships (3D-QSAR) models using CoMFA and CoMSIA analyses. The present study correlates the cell growth inhibition activities of 56 structurally related CEU derivatives to several physicochemical parameters representing steric, electrostatic, and hydrophobic fields. Both CoMFA and CoMSIA models using two different optimum numbers of components (ONC) 10 and 4, respectively, gave good internal predictions and their cross-validated r 2 values were between 0.639 and 0.743. These comprehensive CoMFA and CoMSIA models are useful in understanding the structure–activity relationships of CEU. The two models were compared to the X-ray crystal structure of the complex of tubulin–colchicine and analyzed for similarities between the two modes of analysis. These models will inspire the design of new CEU derivatives with enhanced inhibition of tumor cell growth and targeting specificity of β II-tubulin and the cytoskeleton.

Soluble tubulin complexes, γ-tubulin, and their changing distribution in the zebrafish ( Danio rerio) ovary, oocyte and embryo

Tubulin dynamics, i.e., the interchange of polymeric and soluble forms, is important for microtubule (MTs) cellular functions, and thus plays essential roles in zebrafish oogenesis and embryogenesis. A novel finding in this study revealed that there were soluble pools of tubulins in zebrafish oocytes that were sequestered and maintained in a temporary “oligomeric” state, which retained assembling and disassembling potential (suggested by undetected acetylated tubulin, marker of stable tubulin), but lacked abilities to assemble into MTs spontaneously in vivo. Using differential centrifugation, gel chromatography and DM1A-probed western blot, soluble α-tubulin was found to be associated with large molecular weight complexes (MW range to over 2 MDa) which were reduced in amount by the blastula stage, especially in some batches of embryos, with a concomitant decrease in soluble tubulin. Complexes (MW range less than 2 MDa) then increased in the gastrula with an increase in soluble α-tubulin. Two different anti-γ-tubulin monoclonal antibodies, GTU 88 and TU 30, revealed the existence of soluble γ-tubulin in both zebrafish oocytes and embryos, which also decreased by the blastula stage and increased in the gastrula stage. Soluble α-tubulin and γ-tubulin extracted from zebrafish ovaries, oocytes and embryos co-localized in fractions on three different columns: S-200 Sephacryl, DEAE and Superose-6b. The soluble tubulin complexes were competent to assemble into MTs in vitro induced by taxol, and γ-tubulin was co-localized with assembled MTs. These soluble tubulin complexes were stable during freeze–thaw cycles and resisted high ionic interaction (up to 1.5 M NaCl). Furthermore, some ovarian soluble α-tubulin could be co-immunoprecipitated with γ-tubulin, and vice versa. Two antibodies specific for Xenopus γ-tubulin ring complex proteins (Xgrip 109 and Xgrip 195) detected single bands from ovarian extracts in western blots, suggesting the existence of Xgrip 109 and Xgrip 195 homologues in zebrafish. These findings, together with recent work on γ-tubulin ring complexes in oocytes, eggs and embryos of other species, suggest that soluble γ-tubulin-associated protein complexes may be involved in regulating tubulin dynamics during zebrafish oogenesis and embryogenesis.