Tubulin Dimers Research Articles

Why microtubules run in circles: mechanical hysteresis of the tubulin lattice.

The fate of every eukaryotic cell subtly relies on the exceptional mechanical properties of microtubules. Despite significant efforts, understanding their unusual mechanics remains elusive. One persistent, unresolved mystery is the formation of long-lived arcs and rings, e.g., in kinesin-driven gliding assays. To elucidate their physical origin we develop a model of the inner workings of the microtubule's lattice, based on recent experimental evidence for a conformational switch of the tubulin dimer. We show that the microtubule lattice itself coexists in discrete polymorphic states. Metastable curved states can be induced via a mechanical hysteresis involving torques and forces typical of few molecular motors acting in unison, in agreement with the observations.

Determining a Functional Mechanism for a Dysfunctional Protein

Tau is a microtubule binding protein that forms pathological aggregates in the brain in Alzheimer's disease and other tauopathies. Disease etiology is thought to arise from loss of native interactions between tau and microtubules, as well as from gain of toxicity tied to tau aggregation, although neither mechanism is well-understood. We have investigated the link between function and disease using disease-associated and designed disease-motivated mutants of tau. We find that while the mutants bind microtubules with comparable affinities, they demonstrate an increased affinity for tubulin dimers. Moreover, the mutant forms of tau are impaired in their ability to promote microtubule assembly. Using single molecule FRET, we measure conformational changes in tau upon binding to tubulin that provide a structural framework for the observed altered affinity and function. We propose a model that describes tau binding to tubulin dimers and a mechanism by which disease-relevant alterations to tau impact its function. Together, these results draw attention to the relevance of the interaction between tau and free tubulin as playing an important role in the molecular mechanisms of tau pathology.

Linking microtubules to Parkinson's disease: the case of parkin.

Microtubules (MTs) are dynamic polymers consisting of α/β tubulin dimers and playing a plethora of roles in eukaryotic cells. Looking at neurons, they are key determinants of neuronal polarity, axonal transport and synaptic plasticity. The concept that MT dysfunction can participate in, and perhaps lead to, Parkinson's disease (PD) progression has been suggested by studies using toxin-based and genetic experimental models of the disease. Here, we first learn lessons from MPTP and rotenone as well as from the PD related genes, including SNCA and LRRK2, and then look at old and new evidence regarding the interplay between parkin and MTs. Data from experimental models and human cells point out that parkin regulates MT stability and strengthen the link between MTs and PD paving the way to a viable strategy for the management of the disease.

Development of a novel class of tubulin inhibitor from desmosdumotin B with a hydroxylated bicyclic B-ring.

A series of newly synthesized hydroxylated analogues of triethyldesmosdumotin B (TEDB) with a bicyclic B-ring exhibited a significantly different mode of action for affecting microtubule dynamics and spindle formation but had the same antiproliferative activity spectrum, including activity against multidrug-resistant tumors. These analogues efficiently induced cell cycle arrest at prometaphase and caused formation of immature multipolar spindles. 6'-Hydroxyl TEDB-TB (8) disrupted bipolar spindle formation but had a negligible effect on interphase microtubules. On the basis of the predicted binding modes of the new compounds with tubulin dimer, compound 4 forms three hydrogen bonds (H-bonds) only with α-tubulin at the colchicine site; in contrast, 8 forms H-bonds with both α- and β-tubulin. We predict that, when a compound/ligand, such as 8, forms H-bonds to both α- and β-tubulins, spindle formation is disrupted more than the dynamics of interphase microtubules. This result may reflect the well-known greater dynamicity of spindle microtubules as compared with interphase microtubules.

Decrypting the structural, dynamic, and energetic basis of a monomeric kinesin interacting with a tubulin dimer in three ATPase states by all-atom molecular dynamics simulation.

We have employed molecular dynamics (MD) simulation to investigate, with atomic details, the structural dynamics and energetics of three major ATPase states (ADP, APO, and ATP state) of a human kinesin-1 monomer in complex with a tubulin dimer. Starting from a recently solved crystal structure of ATP-like kinesin-tubulin complex by the Knossow lab, we have used flexible fitting of cryo-electron-microscopy maps to construct new structural models of the kinesin-tubulin complex in APO and ATP state, and then conducted extensive MD simulations (total 400 ns for each state), followed by flexibility analysis, principal component analysis, hydrogen bond analysis, and binding free energy analysis. Our modeling and simulation have revealed key nucleotide-dependent changes in the structure and flexibility of the nucleotide-binding pocket (featuring a highly flexible and open switch I in APO state) and the tubulin-binding site, and allosterically coupled motions driving the APO to ATP transition. In addition, our binding free energy analysis has identified a set of key residues involved in kinesin-tubulin binding. On the basis of our simulation, we have attempted to address several outstanding issues in kinesin study, including the possible roles of β-sheet twist and neck linker docking in regulating nucleotide release and binding, the structural mechanism of ADP release, and possible extension and shortening of α4 helix during the ATPase cycle. This study has provided a comprehensive structural and dynamic picture of kinesin's major ATPase states, and offered promising targets for future mutational and functional studies to investigate the molecular mechanism of kinesin motors.

Decrypting the Structural, Dynamic and Energetic Basis of Kinesin Interacting with Tubulin Dimer in Three ATPase States by All-Atom Molecular Dynamics Simulation

We have employed molecular dynamics (MD) simulation to investigate, with atomic details, the structural dynamics and energetics of three major ATPase states (ADP, APO and ATP state) of a human kinesin 1 complexed with a tubulin dimer. Starting from a recently solved crystal structure of ATP-like kinesin-tubulin complex by Knossow lab, we have performed flexible fitting of cryo-electron-microscopy maps of kinesin-decorated microtubules, followed with extensive MD simulation (400ns for each state), which is to our knowledge the longest MD simulation of kinesin-tubulin complex published to date. Our modeling and simulation have revealed extensive conformational changes at the tubulin-binding site and the nucleotide-binding pocket of kinesin between ADP, APO and ATP state, featuring a more twisted central β-sheet and a highly flexible and open switch I in APO state. We have found kinesin undergoing large structural fluctuations in APO state toward the direction of ATP state, which allosterically couple nucleotide-binding pocket with tubulin-binding site. We have identified a dynamic network of hydrogen bonds spanning the nucleotide-binding pocket and the kinesin-tubulin binding interface, which are predicted to control and couple nucleotide and tubulin binding during kinesin's ATPase cycle. In addition, we have employed binding free energy analysis to identify a set of key residues involved in kinesin-tubulin binding. Our simulation has also shed light on several outstanding issues in kinesin literature (such as the role of neck linker docking in regulating nucleotide binding, and possible extension/shortening of α4 helix during the ATPase cycle). This study has provided a most comprehensive structural and dynamic picture of kinesin's major ATPase states, and offered promising targets for future mutational and functional studies to investigate the molecular mechanism of kinesin motors.

Effect of Lipid Composition on the Affinity and Binding of Dimeric Tubulin to Membranes Studied using Surface Plasmon Resonance, Neutron Reflectivity, Electrophysiology, and AC Electrical Methods

Dimeric tubulin has emerged as an important regulatory factor of the permeability of voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancers. Previously, single-channel studies revealed that the on-rate of the VDAC-tubulin interaction is strongly dependent on the lipid environment. To understand the nature of the binding of this abundant cytosolic water-soluble protein to lipid membranes, we have employed an array of biophysical techniques using unsupported planar lipid membrane and tethered bilayer lipid membrane (tBLM) platforms. Surface plasmon resonance (SPR) of tBLMs shows that tubulin at concentrations less than 100 nM binds irreversibly to DPhPC and 1:1 DOPC:DOPE membranes. The binding rate is significantly larger in high salt concentrations, suggesting a hydrophobic interaction between tubulin and lipid membrane. No binding to DOPC membranes is observed under the same conditions. Electrochemical impedance spectroscopy (EIS) of tBLMs reveals that only DPhPC membranes are strongly perturbed by the binding of tubulin. Neutron reflectivity (NR) measurements on tBLMs give structural information regarding the penetration of the tubulin into the membranes. Electrophysiological measurements of the tubulin/VDAC interaction on the unsupported bilayers confirm the irreversibility of tubulin binding to DPhPC membranes and membranes with significant DOPE content. Second harmonic analysis of planar lipid membranes’ response to ac excitation suggests that tubulin binding causes significant rearrangements of the lipid headgroups. We conclude that tubulin binds to and modifies the structure of lipid membranes even at nanomolar concentrations. Considering that there is up to 10 μM of free dimeric tubulin in cells, our results suggest a new broad regulatory role of dimeric tubulin in vivo.

Isolation of microtubules by assembly/disassembly methods.

The microtubule-isolation procedures described here are based on the ability of the investigator to control the dimer-polymer distribution of tubulin by varying the temperature of the extract. In general, the extract is warmed to induce microtubule assembly, the polymer is collected by centrifugation, cooled to induce disassembly, clarified by centrifugation, and then warmed again to produce polymer. As long as the GTP supply is sufficient, the microtubules that result can be taken through numerous rounds of this in vitro assembly and disassembly reaction. Many microtubule-associated proteins (MAPs) associate with microtubules assembled in vitro. Some reagents can skew the equilibrium of assembly and disassembly toward formation of polymer. The inclusion of glycerol, for instance, promotes microtubule assembly by disrupting the hydration shell around the tubulin dimers. The result is a greater yield of tubulin per gram of starting material. However, the ratio of MAPs to tubulin is slightly lower, presumably because the glycerol also decreases the binding of MAPs to tubulin. Two methods are described here: The first uses buffer lacking assembly-promoting components, and the second uses buffer containing glycerol. These procedures are most efficient with vertebrate brain tissue, where the soluble protein can be up to 15%-20% tubulin. The first produces satisfactory yields when using chick or pig brain; the second is recommended for calf or cow brain. The second procedure may also be useful for studies of nonneuronal tissues where the relative concentration of tubulin per gram of wet weight is considerably lower than that of brain.

Sensitivity of Multiple-Kinesin Transport to Microtubule Lattice Defects

Microtubules are fundamental biopolymers in cells, formed via self-assembly of tubulin dimers. Defects in microtubule lattices have been observed, including point defects (missing tubulin dimers) and line defects (protofilament disruption). Microtubule-based molecular motors enable long-range transport in cells. Potential impact of microtubule lattice defects on intracellular transport is not yet understood. Here we vary microtubule polymerization conditions to uncontrollably tune defect presence in microtubule lattices, and use single-molecule-type optical trapping experiments to investigate the impact of such defect on multiple-kinesin transport. We find that kinesin-based cargoes pause preferentially at specific locations along individual microtubules, and that the pause frequency and duration increase with increasing presence of defects in microtubules. Additionally, we find that the dissociation rates of multiple-kinesin-based cargoes are also strongly dependent on the specific microtubules they travel along. Taken together, our study highlights a previously unexplored, and important role of microtubule lattice assembly in controlling intracellular transport.

High-Affinity Interaction with VDAC Links Cytosolic Proteins to Mitochondrial Regulation in Health, Cancer, and Neurodegeneration

We address the question of how mitochondria control cell survival and cell death by studying the voltage-dependent anion channel (VDAC). VDAC, the major channel of the mitochondrial outer membrane (MOM), is a well-recognized key conduit for ATP and other bioenergetics metabolites fluxes across MOM. We have found that dimeric tubulin, the subunit of microtubule, induces highly efficient reversible blockage of VDAC reconstituted into planar lipid membranes. Although the tubulin-blocked state still conducts small ions, it is virtually impermeable to ATP. We propose that by modulating VDAC permeability for ATP and other respiratory substrates, tubulin controls mitochondrial respiration. These findings are supported by experiments with isolated mitochondria and human hepatoma cells, thus uncovering a mechanism of regulation of mitochondrial energetics by free tubulin and also suggesting how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (the Warburg effect).We also found a functional interaction between VDAC and α-synuclein (α-syn), an intrinsically disordered neuronal protein intimately associated with Parkinson disease (PD) pathogenesis. Importantly, in addition to regulation of VDAC permeability by α-syn, our data indicate that VDAC facilitates translocation of α-syn across MOM where it could target complexes of the mitochondrial respiratory chain in the inner membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. Considering that VDAC is a major conduit for respiratory substrates across the mitochondrial outer membrane, we conclude that the α-syn/VDAC functional interaction reveals the elusive physiological and pathophysiological roles for monomeric α-syn in PD and also in general neurodegeneration.

VDAC3 Forms Typical Voltage-Gated, Anion-Selective, and Tubulin-Sensitive Channels

The Voltage Dependent Anion Channel (VDAC) forms the major pathway for metabolites across the mitochondrial outer membrane (MOM). Mammalian mitochondria have three isoforms of this protein: VDAC1, VDAC2, and VDAC3 which may play different roles in the regulation of mitochondrial functions. VDAC1 is the most studied isoform, since it is the major protein of the MOM in most cells, followed by VDAC2. Both, VDAC1 and VDAC2 reconstituted into planar membranes form voltage-dependent anion-selective channels whose conductance is modulated by dimeric tubulin. As counterpart, the poor channel-forming ability of VDAC3 together with its low proficiency in restoring normal growth in pore-less yeast led to hypothesize that this isoform does not form channels and could be just a modulator of the other two isoforms. We developed an improved protocol of purification and refolding of mouse recombinant VDAC3 that allowed us to fully characterize channels formed by VDAC3 reconstituted in planar lipid membranes and to compare its channel properties with those of VDAC1 and VDAC2. We observed the typical VDAC channel behavior of VDAC3, such as high conductive open state (3.5-4.1 nS in 1M KCl) and multiple low conductive states at potentials ≥ ±40mV, selectivity for anions with PCl-/PK+= 1.7, and typical voltage-gating. This suggests a structural homology of VDAC3 with the other two isoforms. Interestingly, the most distinct characteristic of VDAC3 is its significantly lower sensitivity to the blockage by tubulin, which would lead to an increased permeability of this channel to ATP and other metabolites. This might explain why the down-regulation of VDAC3 expression among the three VDAC isoforms in some cancer cells causes the most drastic decrease in their mitochondrial membrane potential, or why VDAC3 is predominantly expressed in the short-term high energy demanding cells, such as spermatozoa.

Stabilizing versus destabilizing the microtubules: a double-edge sword for an effective cancer treatment option?

Microtubules are dynamic and structural cellular components involved in several cell functions, including cell shape, motility, and intracellular trafficking. In proliferating cells, they are essential components in the division process through the formation of the mitotic spindle. As a result of these functions, tubulin and microtubules are targets for anticancer agents. Microtubule-targeting agents can be divided into two groups: microtubule-stabilizing, and microtubule-destabilizing agents. The former bind to the tubulin polymer and stabilize microtubules, while the latter bind to the tubulin dimers and destabilize microtubules. Alteration of tubulin-microtubule equilibrium determines the disruption of the mitotic spindle, halting the cell cycle at the metaphase-anaphase transition and, eventually, resulting in cell death. Clinical application of earlier microtubule inhibitors, however, unfortunately showed several limits, such as neurological and bone marrow toxicity and the emergence of drug-resistant tumor cells. Here we review several natural and synthetic microtubule-targeting agents, which showed antitumor activity and increased efficacy in comparison to traditional drugs in various preclinical and clinical studies. Cryptophycins, combretastatins, ombrabulin, soblidotin, D-24851, epothilones and discodermolide were used in clinical trials. Some of them showed antiangiogenic and antivascular activity and others showed the ability to overcome multidrug resistance, supporting their possible use in chemotherapy.

Helical alignment inversion of microtubules in accordance with a structural change in their lattice.

Giant helical (oriented chiral nematic) alignments of microtubules of nanometer to centimeter lengths are known to form over a temperature gradient during anisotropic spiral propagation via tubulin dimer addition in a capillary cell. Such helical alignments may be modified by the addition of either paclitaxel or dimethyl sulfoxide, which induces a lattice (helical) structural change in the microtubule itself. In this study, we found that the lattice structural change of microtubules brings about inversion of microtubule alignments in the helical ordering. Based on microscopy and scattering data, a mechanism for the helical ordering of microtubules is discussed in relation to their lattice (helical) structure.

The structure of the complex between α-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism.

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.

Molecular Dynamics Study of the Effect of Polyamine on Microtubule Conformations

Microtubules (MTs) play important roles in cell division, intracellular protein transport, and cell structure and movement in eukaryotic cells. MTs are hollow supramolecular polymers made of alpha and beta tubulin heterodimers. Microtubule assembly in cells require a proper orientation of tubulin molecules to the MTs end. Experiments have shown that polyamines such as spermine can facilitate the diffusion of tubulin dimers in the process of nuclei growth or MT growth. The mechanism of this aggregation is the bridging interaction of polyamines with highly negative C-terminal tail of tubulin surface which helps tubulin together. Recent experiments also show that with a high concentration of spermine, bundles of taxol-stabilized MTs undergo a shape transformation to bundles of inverted tubulin tubules. However, the molecular mechanism underlying the transformation is unclear. We performed all atom molecular dynamics simulation of tubulin clusters in presence of spermine in an explicit water solvent to study the structural dynamics of alpha/beta tubulin and microtubule protofilament. We discuss our results on the interaction between spermine and tubulin proteins and the effect of spermine on conformational dynamics of MT protofilaments.

Shifts in Photon Spectral Power Densities within Schumann (7.7 to 7.8 Hz) Values in Microtubules during Complex Magnetic Field Exposures May Reflect an Information Interface with Universal Energies

The specific diameter of microtubules was shown to be a primary solution when magnetic energy was set equal to Casimir energy. To discern if this spatial containment could be foci for information photon emissions were measured from preparations of microtubules (MTs) while they were exposed in sequential 4 min intervals to various patterns of weak magnetic fields whose intensities ranged from 3 to 10 μT. Calculations from the median mass of a tubulin dimer, its summed charges and the applied magnetic field as well as the change in magnetic moment derived from the energy of the hydrogen line when applied to our experimental fields predicted a dynamic shift (Δf) between 0.03 and 0.21 Hz. Spectral power densities (SPD) indicated marked enhancements in photon numbers during periods of magnetic field exposures within the 7.6 to 7.8 Hz increment. The total SPD units for the shift were 10-18 to 10-17 J per s. Five of the eight patterns elicited a split spectrum of power within this range. Separate factor analyses of the SPDs of the serial values that composed the points of the actual field patterns indicated those that evoked the split-spectrum (Δf = 0.05 to 0.13 Hz) displayed significantly higher loadings on the same factor compared to those that did not. If this shift in photon energy reflects a phase modulation of the coherence frequency (8 MHz) of MTs, the increment of energy per MHz frequency would be within the energy of the neutral hydrogen line. These results suggest that the intrinsic structure or information from specific intensity magnetic fields when applied to MTs is reflected in photon energy densities vacillating around the fundamental Schumann Resonance that could be an interface between Casimir and magnetic sources.

EphrinB1: novel microtubule associated protein whose expression affects taxane sensitivity.

Microtubules (MTs) are components of the cytoskeleton made up of polymerized alpha and beta tubulin dimers. MT structure and function must be maintained throughout the cell cycle to ensure proper execution of mitosis and cellular homeostasis. The protein tyrosine phosphatase, PTPN13, localizes to distinct compartments during mitosis and cytokinesis. We have previously demonstrated that the HPV16 E6 oncoprotein binds PTPN13 and leads to its degradation. Thus, we speculated that HPV infection may affect cellular proliferation by altering the localization of a PTPN13 phosphatase substrate, EphrinB1, during mitosis. Here we report that EphrinB1 co-localizes with MTs during all phases of the cell cycle. Specifically, a cleaved, unphosphorylated EphrinB1 fragment directly binds tubulin, while its phosphorylated form lacks MT binding capacity. These findings suggest that EphrinB1 is a novel microtubule associated protein (MAP). Importantly, we show that in the context of HPV16 E6 expression, EphrinB1 affects taxane response in vitro. We speculate that this reflects PTPN13's modulation of EphrinB1 phosphorylation and suggest that EphrinB1 is an important contributor to taxane sensitivity/resistance phenotypes in epithelial cancers. Thus, HPV infection or functional mutations of PTPN13 in non-viral cancers may predict taxane sensitivity.

Theoretical analysis of microtubule dynamics at all times.

Microtubules are biopolymers consisting of tubulin dimer subunits. As a major component of cytoskeleton they are essential for supporting most important cellular processes such as cell division, signaling, intracellular transport and cell locomotion. The hydrolysis of guanosine triphosphate (GTP) molecules attached to each tubulin subunit supports the nonequilibrium nature of microtubule dynamics. One of the most spectacular properties of microtubules is their dynamic instability when their growth from continuous attachment of tubulin dimers stochastically alternates with periods of shrinking. Despite the critical importance of this process to all cellular activities, its mechanism remains not fully understood. We investigated theoretically microtubule dynamics at all times by analyzing explicitly temporal evolution of various length clusters of unhydrolyzed subunits. It is found that the dynamic behavior of microtubules depends strongly on initial conditions. Our theoretical findings provide a microscopic explanation for recent experiments which found that the frequency of catastrophes increases with the lifetime of microtubules. It is argued that most growing microtubule configurations cannot transit in one step into a shrinking state, leading to a complex overall temporal behavior. Theoretical calculations combined with Monte Carlo computer simulations are also directly compared with experimental observations, and good agreement is found.

New insights into microtubule elongation mechanisms.

Microtubules are cytoskeletal structures in the cytoplasm of eukaryotic cells, and their highly dynamic properties are essential to perform a wide variety of vital functions in cells. Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin molecules. It has largely been assumed that only tubulin dimers can incorporate into microtubules, and that the chemical state of the nucleotide is crucial for the incorporation. Recent observations reveal that both tubulin dimers and oligomers can add to microtubule ends and that the chemical state of the nucleotide is not decisive for tubulin addition. Together with structural studies of tubulin, these results show tubulin assembly polymorphism, which could play a crucial role in microtubule-dependent cellular functions.

Stability Analysis of Jacobi Elliptic Solutions of Microtubule

A nonlinear dynamic model which elucidate the mechanism of the internal motion occuring during the assembly of microtubules (MTs) is considered. The nonlinear space-time dynamics, defined in terms of celebrated ‘solitonic’ equations, brings indispensable tools for understanding, prediction and control of complex behaviors in both physical and life sciences. MTs is assumed to be a single longitudinal degree of freedom per tubulin dimer, and the motion equation is reduced to the discrete nonlinear Schr ¨ odinger equation. Exact solutions are investigated using the Jacobian elliptic fonctions approach. It is shown that such a nonlinear model can lead to the existence of kink solitons, breather as well as Jacobi solutions which can propagate along the microtubule outer surface, and the tubulin tail soliton collisions could serve as elementary computational gates that control cytoskeletal processes. We examine the stability of these solutions and found that the kink solution is stable with respect to small-amplitude fluctuations.