Complexes of tubulin oligomers and tau form a viscoelastic intervening network cross-bridging microtubules into bundles.

The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. Tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in varying concentrations of Mg2+ or Ca2+ divalent cations in mixtures containing αß-tubulin, full-length tau, and GTP at 37 °C in a physiological buffer. A concentration-time kinetic phase diagram generated by synchrotron SAXS reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. SAXS with TEM of plastic-embedded samples provides evidence of a viscoelastic intervening network (IN) of complexes of tubulin oligomers and tau stabilizing MT bundles. In this model, αß-tubulin oligomers in the IN are crosslinked by tau's MT binding repeats, which also link αß-tubulin oligomers to αß-tubulin within the MT lattice. The model challenges whether the cross-bridging of MTs is attributed entirely to MAPs. Tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau, promoting nucleation and growth of tau fibrils in tauopathies.

Regulation of Interfacial Anchoring Orientation of Anisotropic Nanodumbbells.

Nanoparticles exhibiting geometrical and chemical anisotropies hold promise for environmentally responsive materials with tunable mechanical properties. However, a comprehensive understanding of their interfacial behaviors remains elusive. In this paper, we control the interfacial anchoring orientation of polystyrene nanodumbbells by adjusting interparticle forces. The film nanostructure is characterized by the orientation angle analysis of individual dumbbells from cross-sectional EM data: dumbbells undergo orientation transitions from a distinctive horizontal bilayer to an isotropic anchoring when electrostatic repulsion is suppressed by either an ionic strength increase or surface amine-modification. This anchoring orientation influences the film's mechanical properties and foam stability, as investigated by a 2D isotherm and dark/bright-field microscopy measurements. Our findings highlight the potential for precise control of supra-colloidal structures by modulating particle alignment, paving the way for smart delivery systems.

Tubulin Double Helix: Lateral and Longitudinal Curvature Changes of Tubulin Protofilament.

By virtue of their native structures, tubulin dimers are protein building blocks that are naturally preprogrammed to assemble into microtubules (MTs), which are cytoskeletal polymers. Here, polycation-directed (i.e., electrostatically tunable) assembly of tubulins is demonstrated by conformational changes to the tubulin protofilament in longitudinal and lateral directions, creating tubulin double helices and various tubular architectures. Synchrotron small-angle X-ray scattering and transmission electron microscopy reveal a remarkable range of nanoscale assembly structures: single- and double-layered double-helix tubulin tubules. The phase transitions from MTs to the new assemblies are dependent on the size and concentration of polycations. Two characteristic scales that determine the number of observed phases are the size of polycation compared to the size of tubulin (≈4 nm) and to MT diameter (≈25 nm). This work suggests the feasibility of using polycations that have scissor- and glue-like properties to achieve "programmable breakdown" of protein nanotubes, tearing MTs into double-stranded tubulins and building up previously undiscovered nanostructures. Importantly, a new role of tubulins is defined as 2D shape-controllable building blocks for supramolecular architectures. These findings provide insight into the design of protein-based functional materials, for example, as metallization templates for nanoscale electronic devices, molecular screws, and drug delivery vehicles.

Minireview - Microtubules and Tubulin Oligomers: Shape Transitions and Assembly by Intrinsically Disordered Protein Tau and Cationic Biomolecules.

In this minireview, which is part of a special issue in honor of Jacob N. Israelachvili's remarkable research career on intermolecular forces and interfacial science, we present studies of structures, phase behavior, and forces in reaction mixtures of microtubules (MTs) and tubulin oligomers with either intrinsically disordered protein (IDP) Tau, cationic vesicles, or the polyamine spermine (4+). Bare MTs consist of 13 protofilaments (PFs), on average, where each PF is made of a linear stack of αß-tubulin dimers (i.e., tubulin oligomers). We begin with a series of experiments which demonstrate the flexibility of PFs toward shape changes in response to local environmental cues. First, studies show that MT-associated protein (MAP) Tau controls the diameter of microtubules upon binding to the outer surface, implying a shape change in the cross-sectional area of PFs forming the MT perimeter. The diameter of a MT may also be controlled by the charge density of a lipid bilayer membrane that coats the outer surface. We further describe an experimental study where it is unexpectedly found that the biologically relevant polyamine spermine (+4e) is able to depolymerize taxol-stabilized microtubules with efficiency that increases with decreasing temperature. This MT destabilization drives a dynamical structural transition where inside-out curving of PFs, during the depolymerization peeling process, is followed by reassembly of ring-like curved PF building blocks into an array of helical inverted tubulin tubules. We finally turn to a very recent study on pressure-distance measurements in bundles of MTs employing the small-angle X-ray scattering (SAXS)-osmotic pressure technique, which complements the surface-forces-apparatus technique developed by Jacob N. Israelachvili. These latter studies are among the very few which are beginning to shed light on the precise nature of the interactions between MTs mediated by MAP Tau in 37 °C reaction mixtures containing GTP and lacking taxol.

Comparison between 102k and 20k Poly(ethylene oxide) Depletants in Osmotic Pressure Measurements of Interfilament Forces in Cytoskeletal Systems.

The proliferation of successful, cell-free reconstitutions of cytoskeletal networks have prompted measurements of forces between network elements via induced osmotic pressure by the addition of depletants. Indeed, it was through osmotic pressurization that Tau, an intrinsically disordered protein found in neuronal axons, was recently discovered to mediate two distinct microtubule (MT) bundle states, one widely spaced and a second tightly packed, separated by an energy barrier due to polyelectrolyte repulsions between opposing Tau projection domains on neighboring MT surfaces. Here, we compare interfilament force measurements in Tau coated MT bundles using PEO20k (poly(ethylene oxide), Mw = 20000), a commonly used inert depletant, and recently published measurements with PEO102k. While force measurements with either depletant reveals the transition between the two bundled states, measurements with PEO20k cannot recapitulate the correct critical pressure (Pc) at which widely spaced MT bundles transition to tightly packed MT bundles due to depletant penetration into widely spaced bundles below Pc. Surprisingly, upon transitioning to the tightly packed bundle state data from both depletants are in quantitative agreement indicative of expulsion of the smaller PEO20k depletant, but only at distances comparable or less than the PEO20k radius of gyration, significantly smaller than the effective diameter of PEO20k. While PEO102k (with size larger than the wall-to-wall distance between MTs in bundles) can more accurately capture the force response behavior at low to intermediate pressures (<104 Pa), measurements with PEO20k, beyond the overlap regime with PEO102k, extend the achievable osmotic pressure range into the higher-pressure regime (∼5 × 104 Pa). The data underscores the importance of the use of polymeric depletants of different sizes to elucidate force response behavior of cytoskeletal filamentous networks over a more complete extended pressure range.

Synchrotron small-angle X-ray scattering and electron microscopy characterization of structures and forces in microtubule/Tau mixtures.

Tau, a neuronal protein known to bind to microtubules and thereby regulate microtubule dynamic instability, has been shown recently to not only undergo conformational transitions on the microtubule surface as a function of increasing microtubule coverage density (i.e., with increasing molar ratio of Tau to tubulin dimers) but also to mediate higher-order microtubule architectures, mimicking fascicles of microtubules found in the axon initial segment. These discoveries would not have been possible without fine structure characterization of microtubules, with and without applied osmotic pressure through the use of depletants. Herein, we discuss the two primary techniques used to elucidate the structure, phase behavior, and interactions in microtubule/Tau mixtures: transmission electron microscopy and synchrotron small-angle X-ray scattering. While the former is able to provide striking qualitative images of bundle morphologies and vacancies, the latter provides angstrom-level resolution of bundle structures and allows measurements in the presence of in situ probes, such as osmotic depletants. The presented structural characterization methods have been applied both to equilibrium mixtures, where paclitaxel is used to stabilize microtubules, and also to dissipative nonequilibrium mixtures at 37°C in the presence of GTP and lacking paclitaxel.

Paclitaxel suppresses Tau-mediated microtubule bundling in a concentration-dependent manner.

BACKGROUND: Microtubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αß-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius observed for increasing Tau/tubulin-dimer molar ratio ΦTau at paclitaxel/tubulin-dimer molar ratio ΛPtxl=1/1. METHODS: We report a synchrotron SAXS and TEM study of the phase behavior of microtubules as a function of varying concentrations of paclitaxel (1/32≤ΛPtxl≤1/4) and Tau (human isoform 3RS, 0≤Φ3RS≤1/2) at room temperature. RESULTS: Tau and paclitaxel have opposing regulatory effects on microtubule bundling architectures and microtubule diameter. Surprisingly and in contrast to previous results at ΛPtxl=1/1 where microtubule bundles are absent, in the lower paclitaxel concentration regime (ΛPtxl≤1/4), we observe both microtubule doublets and triplets with increasing Tau. Furthermore, increasing paclitaxel concentration (up to ΛPtxl=1/1) slightly decreased the average microtubule diameter (by ~1 PF) while increasing Tau concentration (up to Φ3RS=1/2) significantly increased the diameter (by ~2-3 PFs). CONCLUSIONS: The suppression of Tau-mediated microtubule bundling with increasing paclitaxel is consistent with paclitaxel seeding more, but shorter, microtubules by rapidly exhausting tubulin available for polymerization. Microtubule bundles require the aggregate Tau-Tau attractions along the microtubule length to overcome individual microtubule thermal energies disrupting bundles. GENERAL SIGNIFICANCE: Investigating MAP-mediated interactions between microtubules (as it relates to in vivo behavior) requires the elimination or minimization of paclitaxel.

Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment.

Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent 'active' microtubule bundles at 37 °C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw-w≈25-41 nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw-w≈16-23 nm) is revealed under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau's projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge-charge attractions mediated by weakly penetrating opposing PDs. This landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.

The effect of multivalent cations and Tau on paclitaxel-stabilized microtubule assembly, disassembly, and structure.

In this review we describe recent studies directed at understanding the formation of novel nanoscale assemblies in biological materials systems. In particular, we focus on the effects of multivalent cations, and separately, of microtubule-associated protein (MAP) Tau, on microtubule (MT) ordering (bundling), MT disassembly, and MT structure. Counter-ion directed bundling of paclitaxel-stabilized MTs is a model electrostatic system, which parallels efforts to understand MT bundling by intrinsically disordered proteins (typically biological polyampholytes) expressed in neurons. We describe studies, which reveal an unexpected transition from tightly spaced MT bundles to loose bundles consisting of strings of MTs as the valence of the cationic counter-ion decreases from Z=3 to Z=2. This transition is not predicted by any current theories of polyelectrolytes. Notably, studies of a larger series of divalent counter-ions reveal strong ion specific effects. Divalent counter-ions may either bundle or depolymerize paclitaxel-stabilized MTs. The ion concentration required for depolymerization decreases with increasing atomic number. In a more biologically related system we review synchrotron small angle x-ray scattering (SAXS) studies on the effect of the Tau on the structure of paclitaxel-stabilized MTs. The electrostatic binding of MAP Tau isoforms leads to an increase in the average radius of microtubules with increasing Tau coverage (i.e. a re-distribution of protofilament numbers in MTs). Finally, inspired by MTs as model nanotubes, we briefly describe other more robust lipid-based cylindrical nanostructures, which may have technological applications, for example, in drug encapsulation and delivery.

Surface charge effects on optical trapping of nanometer-sized lipid vesicles.

Optical trapping of nanometer-sized lipid vesicles has been challenging due to the low refractive index contrast of the thin lipid bilayer to the aqueous medium. Using an "optical bottle", a recently developed technique to measure interactions of nanoparticles trapped by an infrared laser, we report, for the first time, quantitative measurements of the trapping energy of charged lipid vesicles. We found that the trapping energy increases with the relative amount of anionic lipids (DOPG) to neutral lipids (DOPC) in vesicles. Moreover, as monovalent salt is added into the exterior solution of vesicles, the trapping energy rapidly approaches zero, and this decrease in trapping energy strongly depends on the amount of anionic lipids in vesicles. A simple model with our experimental observations explains that the trapping energy of charged lipid vesicles is highly correlated with the surface charge density and electric double layer. In addition, we demonstrated selective trapping of a binary mixture of vesicles in different mole fractions of charged lipids, a strategy that has potential implications on charge selective vesicle sorting for engineering applications.

Breathing, crawling, budding, and splitting of a liquid droplet under laser heating.

The manipulation of droplets with sizes on the millimetre scale and below has attracted considerable attention over the past few decades for applications in microfluidics, biology, and chemistry. In this paper, we report the response of an oil droplet floating in an aqueous solution to local laser heating. Depending on the laser power, distinct dynamic transitions of the shape and motion of the droplet are observed, namely, breathing, crawling, budding, and splitting. We found that the selection of the dynamic modes is determined by dynamic instabilities due to the interplay between the convection flows and capillary effects. Our findings can be useful for constructing microfluidic devices to control the motion and shape of a small droplet by simply altering the laser power, and for understanding thermal convective systems with fully soft boundaries.

Spontaneous unilamellar polymer vesicles in aqueous solution.

A unilamellar polymeric vesicle is a self-assembled structure of a block copolymer that forms a spherical single bilayer structure with a hydrophobic interlayer and a hydrophilic surface. Due to their enhanced colloidal stability and mechanical property, controllable surface functionality, or tunable membrane thickness, polymeric vesicles are useful in nano and bio-science, providing potential applications as nanosized carriers for catalysts, drugs, and enzymes. For fabrication of a unilamellar vesicle, however, preparative procedures with a few steps are inherently required. Herein, without complicated preparative procedures, we report spontaneous unilamellar polymeric vesicles with nanometer sizes (<100 nm), which are prepared by simply mixing a triblock copolymer, Pluronic P85 (PEO26PPO40PEO26), and an organic derivative, 5-methyl salicylic acid (5mS), in aqueous solution. Depending on the 5mS concentration and the temperature, the P85-5mS mixtures presented various self-assembled nanostructures such as spherical and cylindrical micelles or vesicles, which were characterized by small angle neutron scattering and cryo-TEM, resulting in a phase diagram drawn as a function of temperature and the 5mS concentration. Interestingly the critical temperature for the micelle-to-vesicle phase transition was easily controlled by varying the 5mS concentration, i.e. it was decreased with increasing the 5mS concentration.

Transformation of taxol-stabilized microtubules into inverted tubulin tubules triggered by a tubulin conformation switch.

Bundles of taxol-stabilized microtubules (MTs)--hollow tubules comprised of assembled αß-tubulin heterodimers--spontaneously assemble above a critical concentration of tetravalent spermine and are stable over long times at room temperature. Here we report that at concentrations of spermine several-fold higher the MT bundles (B(MT)) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (B(ITT)), the outside surface of which corresponds to the inner surface of the B(MT) tubules. Using transmission electron microscopy and synchrotron small-angle X-ray scattering, we quantitatively determined both the nature of the B(MT)-to-B(ITT) transformation pathway, which results from a spermine-triggered conformation switch from straight to curved in the constituent taxol-stabilized tubulin oligomers, and the structure of the B(ITT) phase, which is formed of tubules of helical tubulin oligomers. Inverted tubulin tubules provide a platform for studies requiring exposure and availability of the inside, luminal surface of MTs to MT-targeted drugs and MT-associated proteins.

Simple super-resolution live-cell imaging based on diffusion-assisted Förster resonance energy transfer.

Despite the recent development of several super-resolution fluorescence microscopic techniques, there are still few techniques that can be readily employed in conventional imaging systems. We present a very simple, rapid, general and cost-efficient super-resolution imaging method, which can be directly employed in a simple fluorescent imaging system with general fluorophores. Based on diffusion-assisted Förster resonance energy transfer (FRET), fluorescent donor molecules that label specific target structures can be stochastically quenched by diffusing acceptor molecules, thereby temporally separating otherwise spatially overlapped fluorescence signals and allowing super-resolution imaging. The proposed method provides two- to three-fold-enhancement in spatial resolution, a significant optical sectioning property, and favorable temporal resolution in live-cell imaging. We demonstrate super-resolution live-cell dynamic imaging using general fluorophores in a standard epi-fluorescence microscope with light-emitting diode (LED) illumination. Due to the simplicity of this approach, we expect that the proposed method will prove an attractive option for super-resolution imaging.

Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules.

Microtubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from alpha/beta-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have, shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry (D. J. Needleman et al., Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16099). Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes (I. Koltover et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 14046). The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results which show that, unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (e.g. Ca2+, Sr2+, Ba2+). Instead, many divalent cations pre-empt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca2+ depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the know stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg2+, Mn2+, Co2+, and Zn2+. GdCl3 (3+) is found to be extremely efficient at MT depolymerization requiring ion concentrations of about 1 mM, while oligolysine(2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents either disrupting lateral interactions between PFs (which are strengthened for taxol containing beta-tubulin) or interfering with taxol's ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tublin dimers in a PF).