A critical assessment of the information processing capabilities of neuronal microtubules using coherent excitations

Evidence for signaling, communication, and conductivity in microtubules (MTs) has been shown through both direct and indirect means, and theoretical models predict their potential use in both classical and quantum information processing in neurons. The notion of quantum information processing within neurons has been implicated in the phenomena of consciousness, although controversies have arisen in regards to adverse physiological temperature effects on these capabilities. To investigate the possibility of quantum processes in relation to information processing in MTs, a biophysical MT model is used based on the electrostatic interior of the tubulin protein. The interior is taken to constitute a double-well potential structure within which a mobile electron is considered capable of occupying at least two distinct quantum states. These excitonic states together with MT lattice vibrations determine the state space of individual tubulin dimers within the MT lattice. Tubulin dimers are taken as quantum well structures containing an electron that can exist in either its ground state or first excited state. Following previous models involving the mechanisms of exciton energy propagation, we estimate the strength of exciton and phonon interactions and their effect on the formation and dynamics of coherent exciton domains within MTs. Also, estimates of energy and timescales for excitons, phonons, their interactions, and thermal effects are presented. Our conclusions cast doubt on the possibility of sufficiently long-lived coherent exciton/phonon structures existing at physiological temperatures in the absence of thermal isolation mechanisms. These results are discussed in comparison with previous models based on quantum effects in non-polar hydrophobic regions, which have yet to be disproved.     

Information processing mechanisms in microtubules at physiological temperature: Model predictions for experimental tests

Both direct and indirect experimental evidence has shown signaling, communication and conductivity in microtubules (MTs). Theoretical models have predicted that MTs can be potentially used for both classical and quantum information processing although controversies arose in regard to physiological temperature effects on these capabilities. In this paper, MTs have been studied using well-established principles of classical statistical physics as applied to information processing, information storage and signal propagation. To investigate the existence of information processing in MTs we used cellular automata (CA) models with neighbor rules based on the electrostatic properties of the molecular structure of tubulin, and both synchronous and asynchronous updating methods. We obtained a phase diagram of possible dynamic behaviors in MTs that depend on the values of characteristic physical parameters that can be experimentally verified.     

A nonlinear model of ionic wave propagation along microtubules

Microtubules (MTs) are important cytoskeletal polymers engaged in a number of specific cellular activities including the traffic of organelles using motor proteins, cellular architecture and motility, cell division and a possible participation in information processing within neuronal functioning. How MTs operate and process electrical information is still largely unknown. In this paper we investigate the conditions enabling MTs to act as electrical transmission lines for ion flows along their lengths. We introduce a model in which each tubulin dimer is viewed as an electric element with a capacitive, inductive and resistive characteristics arising due to polyelectrolyte nature of MTs. Based on Kirchhoff’s laws taken in the continuum limit, a nonlinear partial differential equation is derived and analyzed. We demonstrate that it can be used to describe the electrostatic potential coupled to the propagating localized ionic waves. An erratum to this article can be found at     

Cytoskeletal signaling: is memory encoded in microtubule lattices by CaMKII phosphorylation?

Memory is attributed to strengthened synaptic connections among particular brain neurons, yet synaptic membrane components are transient, whereas memories can endure. This suggests synaptic information is encoded and 'hard-wired' elsewhere, e.g. at molecular levels within the post-synaptic neuron. In long-term potentiation (LTP), a cellular and molecular model for memory, post-synaptic calcium ion (Ca2?) flux activates the hexagonal Ca2?-calmodulin dependent kinase II (CaMKII), a dodacameric holoenzyme containing 2 hexagonal sets of 6 kinase domains. Each kinase domain can either phosphorylate substrate proteins, or not (i.e. encoding one bit). Thus each set of extended CaMKII kinases can potentially encode synaptic Ca2? information via phosphorylation as ordered arrays of binary 'bits'. Candidate sites for CaMKII phosphorylation-encoded molecular memory include microtubules (MTs), cylindrical organelles whose surfaces represent a regular lattice with a pattern of hexagonal polymers of the protein tubulin. Using molecular mechanics modeling and electrostatic profiling, we find that spatial dimensions and geometry of the extended CaMKII kinase domains precisely match those of MT hexagonal lattices. This suggests sets of six CaMKII kinase domains phosphorylate hexagonal MT lattice neighborhoods collectively, e.g. conveying synaptic information as ordered arrays of six "bits", and thus "bytes", with 64 to 5,281 possible bit states per CaMKII-MT byte. Signaling and encoding in MTs and other cytoskeletal structures offer rapid, robust solid-state information processing which may reflect a general code for MT-based memory and information processing within neurons and other eukaryotic cells.     

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.     

Structural studies of N-terminal mutants of Connexin 32 using (1)H NMR spectroscopy

Microtubules (MTs) control cell replication, material transport and motion in eukaryotic cells, but MT role in several pathologies is still unknown. These functions are related to the MT physico-chemical properties and MT formation mode starting from tubulin molecules. This study describes a new method, based on the computer aided analysis of the electron paramagnetic resonance (EPR) spectra of selected spin probes to obtain structural and dynamical information on tubulins and MTs and the kinetics of MTs formation promoted by guanosine-5'-triphosphate (GTP). It was found that tubulin and MTs avoid radical quenching caused by ethylene glycol tetraacetic acid (EGTA). MT formation showed different kinetics as a function of tubulin concentration. At 5 mg/mL of tubulin, MTs were formed in 8 min. These results are also useful for getting information on MT-drug interactions.     

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.     

Ideas in Theoretical Biology Do MTS have the Function of Message Transmission?

Structurally, microtubules (MTs) are composed of protofilaments of the subunit protein. They are prominent components of the cytoplasmic matrix and perform important functions as cytoskeletal elements for the determination of cell shape and as key elements in intracellular motility such as mitosis and the translocation of cell organelles. These functions are thought to depend on the controlled assembly and disassembly of MTs in the cytoplasm and on the interaction of MTs with each other and with other cytoplasmic components. I think that apart from these cellular functions, MTs have the function of message transmission. Although no direct evidence is available to explain this point at present, a number of inddirect evidences have been obtained by many scientists e.g.: brain tissue has circumstantial the highest tubulin concentration, MTs have the property of self-assembly and disassembly, microtubule(MT) network is a key factor in differentiation of plant cells.     

Polyglutamylation: a fine‐regulator of protein function?

Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of polyglutamylation and now allows for direct functional studies of the role of tubulin polyglutamylation. Moreover, the recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes.     

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.     

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.     

Root contraction in hyacinth. II. Changes in tubulin levels,microtubule number and orientation associated with differential cell expansion

Root contraction in hyacinth (Hyacinthus orientalis L.) is marked by reoriented cell growth in the cortex of the contractile region. Cellular volume of the inner cortex enlarges fourfold during root contraction. This is associated with large increases in the radial and tangential dimensions and decreases in the longitudinal dimension of the cells. In order to determine the possible role of microtubules (MTs) in these changes we compared tubulin levels and MT numbers and orientation in contracted and non-contracted regions of hyacinth roots. Tubulin content was analysed by a radioimmunoassay; MT numbers and orientation were analyzed by counting profiles in sectioned material using transmission electron microscopy. Contracted tissue was found to have significantly higher levels of tubulin on a per-cell basis than non-contracted tissue, and also increased tubulin levels relative to total protein. The spatial MT frequencies were the same in contracted and non-contracted tissues, indicating a proportional increase in MT numbers in the expanded cells. Although the absolute spatial frequency of MTs was constant, the orientation, as determined by morphometric analysis of MT profiles, was not. While in the longitudinal section plane 42% of the MTs in the non-contracted cells were oblique, in the contracted cells the percentage of MTs presenting oblique profiles increased to 87%. Additionally, a qualitative difference in MTs was observed in contracted cells; electron-opaque material was seen peripherally associated with the MTs of the inner cortex. The changes in tubulin levels and in MT numbers as well as the qualitative differences in the MTs of contracted and non-contracted root regions indicate that, in hyacinth, reoriented cellular enlargement associated with root contraction cannot be explained simply by shifts in the arrangement of preexisting cortical MT arrays, but involves more complex changes in the cytoskeleton.Abbreviations MT(s) microtubule(s) - TEM transmission electron microscopy - RIA radioimmunoassay - M_r apparent molecular mass I=Jernstedt (1984b)     

Microinjection of fluorescent tubulin into plant cells provides a representative picture of the cortical microtubule array

By microinjecting rhodamine-labelled tubulin into living plant cells, it is possible to observe microtubules (MTs) directly and to see how the cortical array reorganizes itself. The validity of the conclusions drawn from such observations depends upon the assumption that most, if not all, of the native MTs are dynamic and incorporate labelled tubulin. However, if arrays also contain MTs that are not exchanging tubulin subunits, such MTs will remain unlabelled, and the labelled MT population will be under-representative of the whole array. To address this potential problem, we microinjected pea epidermal cells with rhodamine-labelled tubulin, then fixed the cells and used fluorescein-conjugated antibodies against tubulin to detect the entire MT array. The two fluorescent patterns corresponded well, confirming that the MTs labelled with exogenous tubulin were evenly distributed throughout the entire array. Also, by comparing the MT image before and after aldehyde fixation, we observed that, although some of the MTs were lost in the procedure, the fixation was able to preserve the arrangement of MTs seen in the living cell. We conclude that fluorescence analogue cytochemistry provides a valid representation of the entire cortical MT array.     

Anisotropic elastic network modeling of entire microtubules

Microtubules are supramolecular structures that make up the cytoskeleton and strongly affect the mechanical properties of the cell. Within the cytoskeleton filaments, the microtubule (MT) exhibits by far the highest bending stiffness. Bending stiffness depends on the mechanical properties and intermolecular interactions of the tubulin dimers (the MT building blocks). Computational molecular modeling has the potential for obtaining quantitative insights into this area. However, to our knowledge, standard molecular modeling techniques, such as molecular dynamics (MD) and normal mode analysis (NMA), are not yet able to simulate large molecular structures like the MTs; in fact, their possibilities are normally limited to much smaller protein complexes. In this work, we developed a multiscale approach by merging the modeling contribution from MD and NMA. In particular, MD simulations were used to refine the molecular conformation and arrangement of the tubulin dimers inside the MT lattice. Subsequently, NMA was used to investigate the vibrational properties of MTs modeled as an elastic network. The coarse-grain model here developed can describe systems of hundreds of interacting tubulin monomers (corresponding to up to 1,000,000 atoms). In particular, we were able to simulate coarse-grain models of entire MTs, with lengths up to 350 nm. A quantitative mechanical investigation was performed; from the bending and stretching modes, we estimated MT macroscopic properties such as bending stiffness, Young modulus, and persistence length, thus allowing a direct comparison with experimental data.     

Characterization and Intracellular Distribution of Microtubule-Associated Protein 2 in Differentiating Human Neuroblastoma Cells

The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.     

A biopolymer transistor: electrical amplification by microtubules

Microtubules (MTs) are important cytoskeletal structures engaged in a number of specific cellular activities, including vesicular traffic, cell cyto-architecture and motility, cell division, and information processing within neuronal processes. MTs have also been implicated in higher neuronal functions, including memory and the emergence of "consciousness". How MTs handle and process electrical information, however, is heretofore unknown. Here we show new electrodynamic properties of MTs. Isolated, taxol-stabilized MTs behave as biomolecular transistors capable of amplifying electrical information. Electrical amplification by MTs can lead to the enhancement of dynamic information, and processivity in neurons can be conceptualized as an "ionic-based" transistor, which may affect, among other known functions, neuronal computational capabilities.     

Compartment volume influences microtubule dynamic instability: a model study

Microtubules (MTs) are cytoskeletal polymers that exhibit dynamic instability, the random alternation between growth and shrinkage. MT dynamic instability plays an essential role in cell development, division, and motility. To investigate dynamic instability, simulation models have been widely used. However, conditions under which the concentration of free tubulin fluctuates as a result of growing or shrinking MTs have not been studied before. Such conditions can arise, for example, in small compartments, such as neuronal growth cones. Here we investigate by means of computational modeling how concentration fluctuations caused by growing and shrinking MTs affect dynamic instability. We show that these fluctuations shorten MT growth and shrinkage times and change their distributions from exponential to non-exponential, gamma-like. Gamma-like distributions of MT growth and shrinkage times, which allow optimal stochastic searching by MTs, have been observed in various cell types and are believed to require structural changes in the MT during growth or shrinkage. Our results, however, show that these distributions can already arise as a result of fluctuations in the concentration of free tubulin due to growing and shrinking MTs. Such fluctuations are possible not only in small compartments but also when tubulin diffusion is slow or when many MTs (de)polymerize synchronously. Volume and all other factors that influence these fluctuations can affect MT dynamic instability and, consequently, the processes that depend on it, such as neuronal growth cone behavior and cell motility in general.     

Modulating Microtubules: A Molecular Perspective on the Effects of Tail Modifications

Microtubules (MTs), an essential component of the eukaryotic cytoskeleton, are a lattice of polymerized tubulin dimers and are crucial for various cellular processes. The genetic and chemical diversity of tubulin and their disordered tails gives rise to a “tubulin code”. The functional role of tubulin post-translational modifications (PTMs), which contribute to the chemical diversity of the tubulin code, is gradually being unraveled. However, variation in the length and spatial organization of tubulin poly-modifications leads to an enormous combinatorial PTM space, which is difficult to study experimentally. Hence, the impact of the combinatorial tubulin PTM space on the biophysical properties of tubulin tails and their interactions with other proteins remains elusive.Here, we combine all-atom and coarse-grained molecular dynamics simulations to elucidate the biophysical implications of the large combinatorial tubulin PTM space in the context of an MT lattice. We find that tail–body interactions are more dominant in the tubulin dimer than in an MT lattice, and are more significant for the tails of α compared with β tubulin. In addition, polyglutamylation, but not polyglycylation, expands the dimensions of the tubulin tails. Polyglutamylation also leads to a decrease in the diffusion rate of MT-associated protein EB1 on MTs, while polyglycylation often increases diffusion rate. These observations are generally not sensitive to the organization of the polymodifications. The effect of PTMs on MT charge density and tail dynamics are also discussed. Overall, this study presents a molecular quantification of the biophysical properties of tubulin tails and their polymodifications, and provides predictions on the functional importance of tubulin PTMs.     

Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein

Oligodendrocytes (OLs) extend arborized processes that are supported by microtubules (MTs) and microfilaments. Little is known about proteins that modulate and interact with the cytoskeleton during myelination. Several lines of evidence suggest a role for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in mediating process formation in OLs. In this study, we report that tubulin is a major CNP-interacting protein. In vitro, CNP binds preferentially to tubulin heterodimers compared with MTs and induces MT assembly by copolymerizing with tubulin. CNP overexpression induces dramatic morphology changes in both glial and nonglial cells, resulting in MT and F-actin reorganization and formation of branched processes. These morphological effects are attributed to CNP MT assembly activity; branched process formation is either substantially reduced or abolished with the expression of loss-of-function mutants. Accordingly, cultured OLs from CNP-deficient mice extend smaller outgrowths with less arborized processes. We propose that CNP is an important component of the cytoskeletal machinery that directs process outgrowth in OLs.