Microtubule Surface Research Articles

Visualizing the Submolecular Organization of αβ-Tubulin Subunits on the Microtubule Inner Surface Using Atomic Force Microscopy.

Microtubules (MTs) are dynamic cytoskeletal polymers essential for mediating fundamental cellular processes, including cell division, intracellular transport, and cell shape maintenance. Understanding the arrangement of tubulin heterodimers within MTs is key to their function. Using frequency modulation atomic force microscopy (FM-AFM) and simulations, we revealed the submolecular arrangement of α- and β-tubulin subunits on the inner MT surface. We observed an undulating molecular arrangement of protofilaments (PFs) with alternating height variations, attributed to different structural orientations and the confirmation of αβ-tubulin heterodimers in adjacent PFs, forming bimodal lateral contacts, as confirmed by AFM simulations. Structural defects resulting from missing tubulin units were directly identified. This detailed structural information provides critical insight into the MT functional properties. Our findings highlight the potential of FM-AFM in liquid as a powerful tool for elucidating the complex interactions among MTs, MT-associated proteins, and other molecules, which are essential for understanding MT dynamics in the cellular context.

Investigation of the expression of Cis P-tau and Pin1 proteins following air pollution induction in the brain tissue of C57BL/6 mice.

Alzheimer's disease (AD) is a multifactorial disease in which environmental factors play a role. Among environmental factors, air pollution is a vital issue in modern life. Despite extensive considerations, it remains uncertain how pollution mediates neurodegeneration in AD. Beta-amyloids and hyperphosphorylated tau proteins are the two main pathological markers that have been studied in AD so far. Tau protein is basically a phosphoprotein whose functions are controlled by phosphorylation. The function of tau protein is to be located on the surface of microtubules and stabilize them. Studies have shown that phosphorylated tau protein (p-tau) exists in cis and trans conformations at Thr231, among which cis is highly neurotoxic. The Pin1 enzyme performs the conversion of cis to trans or vice versa. In this study, an experimental mouse model was designed to investigate the formation of cis p-tau by inducing air pollution. In this way, mice were randomly exposed to pollution at 2-week, 1-month, and 2-month intervals. We investigated the formation of phosphorylated cis tau form during air pollution on mouse brains using Western blots and immunofluorescence. The fluorescent imaging results and Western blotting analysis of mouse brains revealed a significant accumulation of cis p-tau in pollution-treated mice models compared to the healthy control mice. According to Western blot results, air pollution induction caused a significant decrease in Pin1 protein. The results clearly show that the tauopathy observed during air pollution is mediated through the formation of cis tau. Our findings unravel tauopathy mysteries upon pollution and would help find a possible therapeutic target to fight the devastating disorder caused by modern life.

Construction of Silver Nanoparticles inside Microtubules Using Tau-Derived Peptide Ligated with Silver-Binding Peptide

Abstract Silver nanoparticles (AgNPs) and silver nanowires (AgNWs) are interesting nanomaterials that attract significant research attention. The use of peptides/proteins as templates is a promising strategy for constructing uniform metal nanoparticles and nanowires, including AgNPs and AgNWs. In this study, the inner space of microtubules was used to grow AgNPs and AgNWs (or nanowire-like one-dimensional nanoparticle assemblies) using a tandem peptide consisting of our developed Tau-derived peptide that binds to the inner surface of microtubules, and a silver-binding peptide. The incorporation of the peptide into microtubules, stabilization by crosslinking using glutaraldehyde, and subsequent incubation with a silver ion source and reductant, resulted in the formation of uniform AgNPs inside microtubules. The density and morphology of the AgNPs were varied by altering the incubation times and concentrations of the silver ion source and reductant. The developed AgNP-containing microtubules could be useful for future nanotechnological applications, particularly in nanoelectronics and dynamic nanomaterials.

Kinesin-microtubule interaction reveals the mechanism of kinesin-1 for discriminating the binding site on microtubule

Microtubule catalyzes the mechanochemical cycle of kinesin, a kind of molecular motor, through its crucial roles in kinesin’s gating, ATPase and force-generation process. These functions of microtubule are realized through the kinesin-microtubule interaction. The binding site of kinesin on the microtubule surface is fixed. For most of the kinesin-family members, the binding site on microtubule is in the groove between α-tubulin and β-tubulin in a protofilament. The mechanism of kinesin searching for the appropriate binding site on microtubule is still unclear. Using the molecular dynamics simulation method, we investigate the interactions between kinesin-1 and the different binding positions on microtubule. The key non-bonded interactions between the motor domain and tubulins in kinesin’s different nucleotide-binding states are listed. The differences of the amino-acid sequences between α- and β-tubulins make kinesin-1 binding to the α–β groove much more favorable than to the β–α groove. From these results, a two-step mechanism of kinesin-1 to discriminate the correct binding site on microtubule is proposed. Most of the kinesin-family members have the conserved motor domain and bind to the same site on microtubule, the mechanism may also be shared by other family members of kinesin.

Causes, costs and consequences of kinesin motors communicating through the microtubule lattice.

Microtubules are critical for a variety of important functions in eukaryotic cells. During intracellular trafficking, molecular motor proteins of the kinesin superfamily drive the transport of cellular cargoes by stepping processively along the microtubule surface. Traditionally, the microtubule has been viewed as simply a track for kinesin motility. New work is challenging this classic view by showing that kinesin-1 and kinesin-4 proteins can induce conformational changes in tubulin subunits while they are stepping. These conformational changes appear to propagate along the microtubule such that the kinesins can work allosterically through the lattice to influence other proteins on the same track. Thus, the microtubule is a plastic medium through which motors and other microtubule-associated proteins (MAPs) can communicate. Furthermore, stepping kinesin-1 can damage the microtubule lattice. Damage can be repaired by the incorporation of new tubulin subunits, but too much damage leads to microtubule breakage and disassembly. Thus, the addition and loss of tubulin subunits are not restricted to the ends of the microtubule filament but rather, the lattice itself undergoes continuous repair and remodeling. This work leads to a new understanding of how kinesin motors and their microtubule tracks engage in allosteric interactions that are critical for normal cell physiology.

Preparation of a Cellulosic Photosensitive Hydrogel for Tubular Tissue Engineering.

Since the concept of tissue engineering was proposed, biocompatible hydrogel materials have attracted the attention of researchers. With the help of three-dimensional (3D) printing technology, precise shaping of hydrogels can be realized. In this paper, we synthesized a cellulosic photosensitive acrylamide (AM)/N,N-methylenebisacrylamide (MBA) hydrogel. With the high-efficiency water-soluble photoinitiator TPO@Tw developed by our research group, the efficient photocuring cross-linking process of the hydrogel can be realized under 405 nm visible light. In consideration of the viscosity, curing mass, curing depth, and break distance of the hydrogel, we screened out hydroxypropyl cellulose (HPC) as the preferred tackifier of the material. The addition of HPC greatly improved the mechanical properties of the hydrogel. The compressive modulus of the optimal sample AM-HPC-5 increased by 709.2% and the tensile strength increased by 76.7% compared with the blank control group. By adding a PEGDA shell to the surface of the material, the water retention capacity of the hydrogel was effectively improved. The water loss rate was greatly reduced. The 3D wooden-pile structure model was printed by a DIW 3D printer. Further, through coaxial extrusion, the microtubule structure that may be applied in tissue engineering was obtained. Cell experiment results showed high biocompatibility of the hydrogel. NIH 3T3 cells could adhere and grow on the surface of microtubules.

In silico designed microtubule-stabilizer drugs against tauopathy in Alzheimer’s disease

Microtubules are the main building blocks of the cytoskeleton that maintain the shape of the cell. Microtubule-associated proteins, such as Tau protein, facilitate their plasticity in cells. Highly phosphorylated Tau has weak affinity to microtubule and, hence, high probability of aggregation into neurofibrillary tangles (tauopathy). Alzheimer’s disease evolves when Tau proteins are abnormally phosphorylated. To prevent tauopathy in Alzheimer’s disease, we designed drugs de novo targeting them in silico to the phosphorylated Tau-microtubule complexes. Our molecular docking (AutoDock, MOE, GOLD) and molecular dynamics (GROMACS, 2019.6) simulation results revealed compound 23 (C12H28N4O5) as a potential drug candidate, since it can bind (-11.1 kcal/mol by AutoDock) and fix not only phosphorylated Tau on the surface of microtubules, but also prevent their aggregation into bundles. In addition, compound 23 has shown its ability to de-bundle already grouped phosphorylated peptides into single pieces. Communicated by Ramaswamy H. Sarma

Microtubule lattice spacing governs cohesive envelope formation of tau family proteins.

Tau is an intrinsically disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive 'envelopes' around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau envelope disassembly. Investigating other members of the tau family, we find that MAP2 similarly forms envelopes governed by lattice spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct regions. We conclude that the interdependent allostery between lattice spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

Reconstitution of kinetochore motility and microtubule dynamics reveals a role for a kinesin-8 in establishing end-on attachments.

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.

Ciliary central apparatus structure reveals mechanisms of microtubule patterning.

A pair of extensively modified microtubules form the central apparatus (CA) of the axoneme of most motile cilia, where they regulate ciliary motility. The external surfaces of both CA microtubules are patterned asymmetrically with large protein complexes that repeat every 16 or 32 nm. The composition of these projections and the mechanisms that establish asymmetry and longitudinal periodicity are unknown. Here, by determining cryo-EM structures of the CA microtubules, we identify 48 different CA-associated proteins, which in turn reveal mechanisms for asymmetric and periodic protein binding to microtubules. We identify arc-MIPs, a novel class of microtubule inner protein, that bind laterally across protofilaments and remodel tubulin structure and lattice contacts. The binding mechanisms utilized by CA proteins may be generalizable to other microtubule-associated proteins. These structures establish a foundation to elucidate the contributions of individual CA proteins to ciliary motility and ciliopathies.

The pathogenic R5L mutation disrupts formation of Tau complexes on the microtubule by altering local N-terminal structure

The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including progressive supranuclear palsy (PSP), Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that missense mutations in Tau reduce microtubule-binding affinity or increase Tau propensity to aggregate. Here, we investigate the effect of an N-terminal arginine to leucine mutation at position 5 in Tau (R5L), associated with PSP, on Tau-microtubule interactions using an invitro reconstituted system. Contrary to the canonical disease-state theory, we determine that the R5L mutation does not reduce Tau affinity for the microtubule using total internal reflection fluorescence microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger-order complexes, or Tau patches, at high concentrations of Tau. Using NMR, we show that the R5L mutation results in a local structural change that reduces interactions of the projection domain in the presence of microtubules. Altogether, these results challenge both the current paradigm of how mutations in Tau lead to disease and the role of the projection domain in modulating Tau behavior on the microtubule surface.

Elucidating tubulin E-hook peptide structure using spectroscopic approaches

Microtubules (MT) consist of α- and β-tubulin dimers and contain disordered, C-terminal tails, known as E-hooks, which extend from the MT surface to interact with the cytoplasmic environment. While the MT core is conserved, the highly negatively charged E-hook composition differs depending on its location and proposed function, and E-hooks also engage in the binding and processive motility of MT-based motors. Despite the E-hook's clear importance in cytoskeletal regulation and function, its molecular structure is not well understood.

Encapsulation of Nanomaterials Inside Microtubules by Using a Tau-Derived Peptide.

Microtubules (MTs) are tubular cytoskeletons, which are used for the various applications such as active matters and therapeutic targets. Although modification of the exterior surface of MTs is frequently used for functionalization of MTs, there was no approach to introduce molecules inside MTs. We previously developed a unique peptide binding to the inner surface of MT, which is derived from a MT-associated protein, Tau. The Tau-derived peptide (TP) can be used to introduce various nanomaterials inside MTs. Here we describe the TP-based encapsulation of fluorescent dye, gold nanoparticle, green fluorescent protein, and magnetic CoPt nanoparticles inside MTs.

A Nanometric Probe of the Local Proton Concentration in Microtubule-Based Biophysical Systems.

We show a double-functional fluorescence sensing paradigm that can retrieve nanometric pH information on biological structures. We use this method to measure the extent of protonic condensation around microtubules, which are protein polymers that play many roles crucial to cell function. While microtubules are believed to have a profound impact on the local cytoplasmic pH, this has been hard to show experimentally due to the limitations of conventional sensing techniques. We show that subtle changes in the local electrochemical surroundings cause a double-functional sensor to transform its spectrum, thus allowing a direct measurement of the protonic concentration at the microtubule surface. Microtubules concentrate protons by as much as one unit on the pH scale, indicating a charge storage role within the cell via the localized ionic condensation. These results confirm the bioelectrical significance of microtubules and reveal a sensing concept that can deliver localized biochemical information on intracellular structures.

Augmin deficiency in neural stem cells causes p53-dependent apoptosis and aborts brain development

Microtubules that assemble the mitotic spindle are generated by centrosomal nucleation, chromatin-mediated nucleation, and nucleation from the surface of other microtubules mediated by the augmin complex. Impairment of centrosomal nucleation in apical progenitors of the developing mouse brain induces p53-dependent apoptosis and causes non-lethal microcephaly. Whether disruption of non-centrosomal nucleation has similar effects is unclear. Here, we show, using mouse embryos, that conditional knockout of the augmin subunit Haus6 in apical progenitors led to spindle defects and mitotic delay. This triggered massive apoptosis and abortion of brain development. Co-deletion of Trp53 rescued cell death, but surviving progenitors failed to organize a pseudostratified epithelium, and brain development still failed. This could be explained by exacerbated mitotic errors and resulting chromosomal defects including increased DNA damage. Thus, in contrast to centrosomes, augmin is crucial for apical progenitor mitosis, and, even in the absence of p53, for progression of brain development.

Fast Leaps between Millisecond Confinements Govern Ase1 Diffusion along Microtubules

Diffusion is the most fundamental mode of protein translocation within cells. Confined diffusion of proteins along the electrostatic potential constituted by the surface of microtubules, although modeled meticulously in molecular dynamics simulations, has not been experimentally observed in real-time. Here, interferometric scattering microscopy is used to directly visualize the movement of the microtubule-associated protein Ase1 along the microtubule surface at nanometer and microsecond resolution. Millisecond confinements of Ase1 and fast leaps between these positions of dwelling preferentially occurring along the microtubule protofilaments are resolved, revealing Ase1's mode of diffusive translocation along the microtubule's periodic surface. The derived interaction potential closely matches the tubulin-dimer periodicity and the distribution of the electrostatic potential on the microtubule lattice. It is anticipated that mapping the interaction landscapes for different proteins on microtubules, finding plausible energetic barriers of different positioning and heights, can provide valuable insights into regulating the dynamics of essential cytoskeletal processes, such as intracellular cargo trafficking, cell division, and morphogenesis, all of which rely on diffusive translocation of proteins along microtubules.

Tracking the Amide I and αCOO- Terminal ν(C=O) Raman Bands in a Family of l-Glutamic Acid-Containing Peptide Fragments: A Raman and DFT Study.

The E-hook of β-tubulin plays instrumental roles in cytoskeletal regulation and function. The last six C-terminal residues of the βII isotype, a peptide of amino acid sequence EGEDEA, extend from the microtubule surface and have eluded characterization with classic X-ray crystallographic techniques. The band position of the characteristic amide I vibration of small peptide fragments is heavily dependent on the length of the peptide chain, the extent of intramolecular hydrogen bonding, and the overall polarity of the fragment. The dependence of the E residue’s amide I ν(C=O) and the αCOO− terminal ν(C=O) bands on the neighboring side chain, the length of the peptide fragment, and the extent of intramolecular hydrogen bonding in the structure are investigated here via the EGEDEA peptide. The hexapeptide is broken down into fragments increasing in size from dipeptides to hexapeptides, including EG, ED, EA, EGE, EDE, DEA, EGED, EDEA, EGEDE, GEDEA, and, finally, EGEDEA, which are investigated with experimental Raman spectroscopy and density functional theory (DFT) computations to model the zwitterionic crystalline solids (in vacuo). The molecular geometries and Boltzmann sum of the simulated Raman spectra for a set of energetic minima corresponding to each peptide fragment are computed with full geometry optimizations and corresponding harmonic vibrational frequency computations at the B3LYP/6-311++G(2df,2pd) level of theory. In absence of the crystal structure, geometry sampling is performed to approximate solid phase behavior. Natural bond order (NBO) analyses are performed on each energetic minimum to quantify the magnitude of the intramolecular hydrogen bonds. The extent of the intramolecular charge transfer is dependent on the overall polarity of the fragment considered, with larger and more polar fragments exhibiting the greatest extent of intramolecular charge transfer. A steady blue shift arises when considering the amide I band position moving linearly from ED to EDE to EDEA to GEDEA and, finally, to EGEDEA. However, little variation is observed in the αCOO− ν(C=O) band position in this family of fragments.

Microtubules as One-Dimensional Crystals: Is Crystal-Like Structure the Key to the Information Processing of Living Systems?

Each tubulin protein molecule on the cylindrical surface of a microtubule, a fundamental element of the cytoskeleton, acts as a unit cell of a crystal sensor. Electromagnetic sensing enables the 2D surface of microtubule to act as a crystal or a collective electromagnetic signal processing system. We propose a model in which each tubulin dimer acts as the period of a one-dimensional crystal with effective electrical impedance related to its molecular structure. Based on the mathematical crystal theory with one-dimensional translational symmetry, we simulated the electrical transport properties of the signal across the microtubule length and compared it to our single microtubule experimental results. The agreement between theory and experiment suggests that one of the most essential components of any Eukaryotic cell acts as a one-dimensional crystal.

Novel Effect of Tau N-Terminal Mutation, R5L, on Microtubule Mediated Condensation

Understanding the physiological function of the intrinsically disordered microtubule associated protein (MAP) Tau has proven challenging. Tau, primarily expressed in neurons, is known to have a variety of axonal functions, including regulation of microtubule dynamics and modulation of kinesin motor motility. Interestingly, research on the microtubule binding behavior of Tau reveals that Tau binds to the microtubule surface in a dynamic equilibrium between static and diffusive states. Although it is understood that the binding state equilibrium plays a role in physiological Tau function, such as modulation of kinesin motility, it is less understood how disease associated mutations affect Tau binding behavior and function. The canonical theory states that mutations in Tau reduce Tau affinity for the microtubule. Here, we investigate the role of an N-terminal disease associated mutation in Tau, R5L, using an in vitro reconstituted system for single molecule Total Internal Reflection Fluorescence (smTIRF) Microscopy. Contrary to the canonical theory, we determined the R5L mutation does not reduce Tau affinity for the microtubule. Rather, the R5L mutation reduces the total amount of Tau bound to the microtubule at saturating conditions. Our data suggests the R5L mutation reduces Tau:Tau interactions, decreasing the ability of R5L-Tau to form larger order complexes, known as “Tau condensates.” Currently, we are determining the mechanism by which this occurs, potentially due to a structural change associated with the R5L mutation. Altogether, these results challenge the current paradigm of how mutations in Tau lead to disease.

Live-Cell Fluorescence Imaging of Microtubules by Using a Tau-Derived Peptide.

Microtubules (MTs) are important targets for imaging in living cells because of their vital roles in cellular processes. The dynamics (polymerization/depolymerization) of MTs has been imaged in living cells by utilizing MT-targeted drugs as scaffolds. We previously developed a unique MT-binding motif derived from a MT-associated protein, Tau. The Tau-derived peptide (TP) binds to the inner surface of MTs without inhibiting the dynamics of MTs. We introduce a new protocol for live-cell imaging of MTs by using fluorescently labeled TP. We exemplify that tetramethylrhodamine (TMR)-labeled TP (TP-TMR) is spontaneously internalized into HepG2 cells and binds to intracellular MTs, enabling visualization of MTs in living cells. TP-TMR shows no apparent effects on polymerization/depolymerization of MTs and no cytotoxicity. Thus, the peptide-based approach is useful for long-term imaging of MTs.