Tubulin Dimers Research Articles

The role of microtubules in secretory protein transport.

Microtubules are part of the dynamic cytoskeleton network and composed of tubulin dimers. They are the main tracks used in cells to organize organelle positioning and trafficking of cargos. In this Review, we compile recent findings on the involvement of microtubules in anterograde protein transport. First, we highlight the importance of microtubules in organelle positioning. Second, we discuss the involvement of microtubules within different trafficking steps, in particular between the endoplasmic reticulum and the Golgi complex, traffic through the Golgi complex itself and in post-Golgi processes. A large number of studies have assessed the involvement of microtubules in transport of cargo from the Golgi complex to the cell surface. We focus here on the role of kinesin motor proteins and protein interactions in post-Golgi transport, as well as the impact of tubulin post-translational modifications. Last, in light of recent findings, we highlight the role microtubules have in exocytosis, the final step of secretory protein transport, occurring close to focal adhesions.

A Theoretical Study on the Biophysical Mechanisms by Which Tumor Treating Fields Affect Tumor Cells During Mitosis.

A theoretical study on the mechanisms through which Tumor Treating Fields (TTFields) affect dividing tumor cells. Numerical analysis was used to revisit two previously proposed mechanisms and introduce a third. We examine the previous hypotheses that: a) TTFields generate a moment that affects microtubule assembly during early mitosis, and b) dielectrophoretic (DEP) forces cause neutral particles to move toward the cleavage furrow during the telophase stage. We further introduce a new hypothesis that TTFields modify cell membrane potential in dividing tumor cells. a) The Brownian energy is several orders of magnitude larger than the moment induced by TTFields on tubulin dimers. b) Adding Stokes drag forces to DEP forces shows that the motion of the particles in the cytoplasm is very slow, approximately 0.003 µm/s, and therefore, unless the duration of the telophase is long enough there will be no substantial effect from the DEP forces. c) The Schwan equation shows that electric fields at the frequencies of clinical TTFields can cause a 10%-17% change in tumor cell membrane potential. Our studies find limited support for the previously suggested hypotheses and suggest that the TTFields affect ion channels by inducing cell membrane potential change could be a mechanism of tumor cell death. Previously suggested mechanisms of tumor cell death from TTFields are found lacking. The effect of TTFields on the tumor cell membrane potential warrants further research.

Tryptophan Improves Memory Independent of Its Role as a Serotonin Precursor: Potential Involvement of Microtubule Proteins.

There are numerous studies examining the effects of tryptophan on behavioral processes, including learning and memory. While most studies suggest that fluctuations in tryptophan levels exert their effects through modifications in serotonergic neurotransmission, there are other neural mechanisms that have accounted for the observed outcomes as well. In this study, we demonstrated that acute administration of tryptophan modulates spatial and object-recognition memory independent of its role as a serotonin precursor. One possible explanation for the observed improvement in memory is through the interaction between tryptophan and microtubule proteins. Microtubules are key components involved in the morphological and functional development of neurons. Moreover, several models suggest that microtubule dynamics contributes to neural network connectivity, information processing, and memory storage. Here, we examined the interaction between tryptophan and microtubules and indicated that tryptophan is capable of a creating a static interaction with the tubulin dimer through a single binding site. This interaction induces the rate of tubulin assembly and as a result increases polymer mass.

Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex.

Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of ∼2 μM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) chloride (RPC2) retards or arrests microtubule motion as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins. Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase, suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy, these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an associative binding constant of 4.8 × 106 M−1 for RPC2 to preformed microtubules and support a 1 : 1 RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is co-localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule stabilizing agent and sharing many similarities with cells treated with paclitaxel.

In Vitro Reconstitution Assays of Microtubule Amplification and Lattice Repair by the Microtubule-Severing Enzymes Katanin and Spastin.

Microtubules are non-covalent dynamic polymers essential for the life of all eukaryotic cells. Their dynamic behavior is regulated by a large array of cellular effectors. In vitro microtubule assays have been instrumental in dissecting the mechanism of microtubule-associated proteins. In this chapter, we focus on microtubule-severing enzymes katanin and spastin. They are AAA ATPases that generate internal breaks in microtubules by extracting tubulin dimers out of the microtubule lattice. We present protocols for TIRF microscopy-based assays that were instrumental in proving that these enzymes not only sever microtubules but also remodel the microtubule lattice by promoting the exchange of lattice GDP-tubulin with GTP-tubulin from the soluble pool. This activity can modulate microtubule dynamics and support microtubule-dependent microtubule amplification in the absence of a nucleating factor.

Taxanes convert regions of perturbed microtubule growth into rescue sites.

Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anticancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state, and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization.

Katanin Grips the β-Tubulin Tail through an Electropositive Double Spiral to Sever Microtubules.

The AAA ATPase katanin severs microtubules. It is critical in cell division, centriole biogenesis, and neuronal morphogenesis. Its mutation causes microcephaly. The microtubule templates katanin hexamerization and activates its ATPase. The structural basis for these activities and how they lead to severing is unknown. Here, we show that β-tubulin tails are necessary and sufficient for severing. Cryoelectron microscopy (cryo-EM) structures reveal the essential tubulin tail glutamates gripped by a double spiral of electropositive loops lining the katanin central pore. Each spiral couples allosterically to the ATPase and binds alternating, successive substrate residues, with consecutive residues coordinated by adjacent protomers. This tightly couples tail binding, hexamerization, and ATPase activation. Hexamer structures in different states suggest an ATPase-driven, ratchet-like translocation of the tubulin tail through the pore. A disordered region outside the AAA core anchors katanin to the microtubule while the AAA motor exerts the forces that extract tubulin dimers and sever the microtubule.

Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins

Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the Tetrahymena doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a-tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.

Comparison of polymerization and structural behavior of microtubules in rat brain and sperm affected by the extremely low-frequency electromagnetic field

BackgroundMicrotubule proteins are able to produce electromagnetic fields and have an important role in memory formation, and learning. Therefore, microtubules have the potential to be affected by exogenous electromagnetic fields. This study aimed to examine the comparison of microtubule polymerization and its structural behavior in brain and sperm affected by 50 Hz extremely low-frequency electromagnetic field (ELEF).ResultsTwenties adult male rats were randomly and equally divided into control and experimental groups, to evaluate the effect of 50 Hz ELEF on the sperm and brain functions. Plus-maze, serum testosterone and corticosterone, and sperm evaluation were performed. Next, the semen and brain samples were obtained, and they were divided into four experimental groups for investigation of microtubule polymerization. There was no significant difference in testosterone and, corticosterone levels, anxiety behaviors, and sperm morphology between control and ELEF-exposure groups. The sperm viability, total and progressive motility were significantly higher in the ELEF-exposed group than that of the control group. The microtubule polymerization in sperm ELEF was significantly higher than in other groups. The secondary and tertiary structures of tubulins were significantly affected in the brain, and sperm ELEF groups.ConclusionIt seems that the polymerization of microtubules and conformational changes of tubulin dimers are improved by ELEF application.

Dynamical decoration of stabilized-microtubules by Tau-proteins

Tau is a microtubule-associated protein that regulates axonal transport, stabilizes and spatially organizes microtubules in parallel networks. The Tau-microtubule pair is crucial for maintaining the architecture and integrity of axons. Therefore, it is essential to understand how these two entities interact to ensure and modulate the normal axonal functions. Based on evidence from several published experiments, we have developed a two-dimensional model that describes the interaction between a population of Tau proteins and a stabilized microtubule at the scale of the tubulin dimers (binding sites) as an adsorption-desorption dynamical process in which Tau can bind on the microtubule outer surface via two distinct modes: a longitudinal (along a protofilament) and lateral (across adjacent protofilaments) modes. Such a process yields a dynamical distribution of Tau molecules on the microtubule surface referred to as microtubule decoration that we have characterized at the equilibrium using two observables: the total microtubule surface coverage with Tau’s and the distribution of nearest neighbors Tau’s. Using both analytical and numerical approaches, we have derived expressions and computed these observables as a function of key parameters controlling the binding reaction: the stoichiometries of the Taus in the two binding modes, the associated dissociation constants and the ratio of the Tau concentration to that of microtubule tubulin dimers.

Multiscale Computational Modeling of Tubulin-Tubulin Lateral Interaction

Microtubules are multistranded polymers in eukaryotic cells that support key cellular functions such as chromosome segregation, motor-based cargo transport, and maintenance of cell polarity. Microtubules self-assemble via “dynamic instability,” in which the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e., what is different between the GTP- and GDP-tubulin states that enables microtubule growth and shortening, respectively? More generally, a major challenge in biology is how to connect theoretical frameworks across length- and timescales, from atoms to cellular behavior. In this study, we describe a multiscale model by linking atomistic molecular dynamics (MD), molecular Brownian dynamics (BD), and cellular-level thermokinetic modeling of microtubules. Here, we investigated the underlying interaction energy when tubulin dimers associate laterally by performing all-atom MD simulations. We found that the lateral potential energy is not significantly different among three nucleotide states of tubulin, GTP, GDP, and GMPCPP and is estimated to be ≅ −11 kBT. Furthermore, using MD potential energy in our BD simulations of tubulin dimers confirms that the lateral bond is weak on its own, with a mean lifetime of ∼0.1 μs, implying that the longitudinal bond is required for microtubule assembly. We conclude that nucleotide-dependent lateral-bond strength is not the key mediator microtubule dynamic instability, implying that GTP acts elsewhere to exert its stabilizing influence on microtubule polymer. Furthermore, the estimated lateral-bond strength (ΔGlat0≅ −5 kBT) is well-aligned with earlier estimates based on thermokinetic modeling and light microscopy measurements. Thus, we have computationally connected atomistic-level structural information, obtained by cryo-electron microscopy, to cellular-scale microtubule assembly dynamics using a combination of MD, BD, and thermokinetic models to bridge from Ångstroms to micrometers and from femtoseconds to minutes.

Molecular understanding of label-free second harmonic imaging of microtubules

Microtubules are a vital component of the cell’s cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.

Abstract OR-5: Prediction of Relaxed Conformations for Tubulin Dimers: from Cryo- Electron Microscopy to Molecular Dynamics

Abstract OR-5: Prediction of Relaxed Conformations for Tubulin Dimers: from Cryo- Electron Microscopy to Molecular Dynamics

Synthesis, Structure, Stability, and Inhibition of Tubulin Polymerization by RuII–p-Cymene Complexes of Trimethoxyaniline-Based Schiff Bases

Four trimethoxy- and dimethoxyphenylamine-based Schiff base (L1-L4)-bearing RuII-p-cymene complexes (1-4) of the chemical formula [RuII(η6-p-cymene)(L)(Cl)] were synthesized, isolated in pure form, and structurally characterized using single-crystal X-ray diffraction and other analytical techniques. The complexes showed excellent in vitro antiproliferative activity against various forms of cancer that are difficult to cure, viz., triple negative human metastatic breast carcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. The 1H nuclear magnetic resonance data in the presence of 10% dimethylformamide-d7 or dimethyl sulfoxide-d6 in phosphate buffer (pD 7.4, containing 4 mM NaCl) showed that the complexes immediately generate the aquated species that is stable for at least 24 h. Electrospray ionization mass spectrometry data showed that they do not bind with guanine nitrogen even in the presence of 5 molar equivalents of 9-EtG, during a period of 24 h. The best complex in the series, 1, exhibits an IC50 of approximately 10-15 μM in the panel of tested cancer cell lines. The complexes do not enhance the production of reactive oxygen species in the cells. Docking studies with a tubulin crystal structure (Protein Data Bank entry 1SAO ) revealed that 1 and 3 as well as L1 and L3 have a high affinity for the interface of the α and β tubulin dimer in the colchicine binding site. The immunofluorescence studies showed that 1 and 3 strongly inhibited microtubule network formation in MDA-MB-231 cells after treatment with an IC20 or IC50 dose for 12 h. The cell cycle analysis upon treatment with 1 showed that the complexes inhibit the mitotic phase because the arrest was observed in the G2/M phase. In summary, 1 and 3 are RuII half-sandwich complexes that are capable of disrupting a microtubule network in a dose-dependent manner. They depolarize the mitochondria, arrest the cell cycle in the G2/M phase, and kill the cells by an apoptotic pathway.

Lattice defects induce microtubule self-renewal.

Microtubules are dynamic polymers, which grow and shrink by addition and removal of tubulin dimers at their extremities. Within the microtubule shaft, dimers adopt a densely packed and highly ordered crystal-like lattice structure, which is generally not considered to be dynamic. Here we report that thermal forces are sufficient to remodel the microtubule shaft, despite its apparent stability. Our combined experimental data and numerical simulations on lattice dynamics and structure suggest that dimers can spontaneously leave and be incorporated into the lattice at structural defects. We propose a model mechanism, where the lattice dynamics is initiated via a passive breathing mechanism at dislocations, which are frequent in rapidly growing microtubules. These results show that we may need to extend the concept of dissipative dynamics, previously established for microtubule extremities, to the entire shaft, instead of considering it as a passive material.

Tubulin-VDAC Interaction: Molecular Basis for Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy.

Tubulin is a well-established target of microtubule-targeting agents (MTAs), a widely used class of chemotherapeutic drugs. Yet, aside from their powerful anti-cancer efficiency, MTAs induce a dose-limiting and debilitating peripheral neurotoxicity. Despite intensive efforts in the development of neuroprotective agents, there are currently no approved therapies to effectively manage chemotherapy-induced peripheral neuropathy (CIPN). Over the last decade, attempts to unravel the pathomechanisms underlying the development of CIPN led to the observation that mitochondrial dysfunctions stand as a common feature associated with axonal degeneration. Concomitantly, mitochondria emerged as crucial players in the anti-cancer efficiency of MTAs. The findings that free dimeric tubulin could be associated with mitochondrial membranes and interact directly with the voltage-dependent anion channels (VDACs) located in the mitochondrial outer membrane strongly suggested the existence of an interplay between both subcellular compartments. The biological relevance of the interaction between tubulin and VDAC came from subsequent in vitro studies, which found dimeric tubulin to be a potent modulator of VDAC and ultimately of mitochondrial membrane permeability to respiratory substrates. Therefore, one of the hypothetic mechanisms of CIPN implies that MTAs, by binding directly to the tubulin associated with VDAC, interferes with mitochondrial function in the peripheral nervous system. We review here the foundations of this hypothesis and discuss them in light of the current knowledge. A focus is set on the molecular mechanisms behind MTA interference with dimeric tubulin and VDAC interaction, the potential relevance of tubulin isotypes and availability as a free dimer in the specific context of MTA-induced CIPN. We further highlight the emerging interest for VDAC and its interacting partners as a promising therapeutic target in neurodegeneration.

Cages Driven Away from Equilibrium Binding by Electric Fields

Molecules and materials that are pushed away from equilibrium can produce unique dynamic and adaptive properties that cannot be attained when they are at rest. In this issue of Chem, Cockroft and co-workers study the transient binding and debinding of coordination cages to the nanopocket of α-hemolysin and show that the binding and debinding events are driven out of equilibrium by an external electric field. Strong applied fields can invert the cage-nanopore binding selectivities and can enhance enantio-inversion to enrich one chiral form of the cage over the other.

Tau repeat regions contain conserved histidine residues that modulate microtubule-binding in response to changes in pH

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (<7.5), these histidines are positively charged and interact with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH (>7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions.

Molecular modeling study on the differential microtubule-stabilizing effect in singly- and doubly-bonded complexes with peloruside A and paclitaxel.

Microtubules (MT) are dynamic cytoskeletal components that play a crucial role in cell division. Disrupting MT dynamics by MT stabilizers is a widely employed strategy to control cell proliferation in cancer therapy. Most MT stabilizers bind to the taxol (TX) site located at the luminal interface between protofilaments, except laulimalide and peloruside A (PLA), which bind to an interfacial pocket on outer MT surface. Cryo-electron microscopy MTs reconstructions have shown differential structural effects on the MT lattice in singly- and doubly-bonded complexes with PLA, TX, and PLA/TX, as PLA is able to revert the lattice heterogeneity induced by TX association leading to more regular MT assemblies. In this work, fully-atomistic molecular dynamics simulations were employed to examine the single and double association of MT stabilizers to reduced MT models in the search for structural and energetic evidence that could be related to the differential regularization and stabilization effects exerted by PLA and TX on the MT lattice. Our results revealed that the double association of PLA/TX (a) strengthens the lateral contact between tubulin dimers compared to singly-bonded complexes, (b) favors a more parallel arrangement between tubulin dimers, and (c) induces a larger restriction in the interdimeric conformational motion increasing the probability of finding structures consistent with 13-protofilaments arrangements. These results and are valuable to increase understanding about the molecular mechanism of action of MT stabilizers, and could account for an overstabilization of MTs in doubly-bonded complexes compared to singly-bonded systems.

Long-range, through-lattice coupling improves predictions of microtubule catastrophe.

Microtubules are cylindrical polymers of αβ-tubulin that play critical roles in fundamental processes such as chromosome segregation and vesicular transport. Microtubules display dynamic instability, switching stochastically between growth and rapid shrinking as a consequence of GTPase activity in the lattice. The molecular mechanisms behind microtubule catastrophe, the switch from growth to rapid shrinking, remain poorly defined. Indeed, two-state stochastic models that seek to describe microtubule dynamics purely in terms of the biochemical properties of GTP- and GDP-bound αβ-tubulin predict the concentration dependence of microtubule catastrophe incorrectly. Recent studies provide evidence for three distinct conformations of αβ-tubulin in the lattice that likely correspond to GTP, GDP.Pi, and GDP. The incommensurate lattices observed for these different conformations raise the possibility that in a mixed nucleotide state lattice, neighboring tubulin dimers might modulate each other’s conformations and hence each other’s biochemistry. We explored whether incorporating a GDP.Pi state or the likely effects of conformational accommodation can improve predictions of catastrophe. Adding a GDP.Pi intermediate did not improve the model. In contrast, adding neighbor-dependent modulation of tubulin biochemistry improved predictions of catastrophe. Because this conformational accommodation should propagate beyond nearest-neighbor contacts, our modeling suggests that long-range, through-lattice effects are important determinants of microtubule catastrophe.