Peter J. Chung

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules.

Significance The microtubule-associated protein Tau is known to stabilize microtubules against depolymerization in neuronal axons, ensuring proper trafficking of organelles along microtubules in long axons. Abnormal interactions between Tau and microtubules are implicated in Alzheimer’s disease and other neurodegenerative disorders. We directly measured forces between microtubules coated with Tau isoforms by synchrotron small-angle X-ray scattering of reconstituted Tau–microtubule mixtures under osmotic pressure (mimicking molecular crowding in cells). We found that select Tau isoforms fundamentally alter forces between microtubules by undergoing a conformational change on microtubule surfaces at a coverage indicative of an unusually extended Tau state. This gain of function by longer isoforms in imparting steric stabilization to microtubules is essential in preventing microtubule aggregation and loss of function in organelle trafficking. Microtubules (MTs) are hollow cytoskeletal filaments assembled from αβ-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer’s. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (P). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; ΦTau = 1/10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to PB ∼ 10,000–20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (PB ∼ 1,000–2,000 Pa) over the same range. Remarkably, the abrupt increase in PB observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < ΦTau < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT–MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment.

Neurofilament networks: Salt-responsive hydrogels with sidearm-dependent phase behavior

BACKGROUND Neurofilaments (NFs) - the neuron-specific intermediate filament proteins - are assembled into 10nm wide filaments in a tightly controlled ratio of three different monomer types: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H). Previous work on reconstituted bovine NF hydrogels has shown the dependence of network properties, including filament alignment and spacing, on the subunit composition. METHODS We use polarized optical microscopy and SAXS to explore the full salt-dependent phase behavior of reconstituted bovine NF networks as a function of various binary and ternary subunit ratios. RESULTS We observe three salt-induced liquid crystalline phases: the liquid-ordered B(G) and N(G) phases, and the disordered I(G) phase. We note the emergent sidearm roles, particularly that of NF-H in driving the parallel to cross-filament transition, and the counter-role of NF-M in suppressing the I(G) phase. CONCLUSIONS In copolymers of NF-LH, NF-H shifts the I(G) to N(G) transition to nearer physiological salt concentrations, as compared to NF-M in copolymers of NF-LM. For ternary mixtures, the role of NF-H is modulated by the ratio of NF-M, where beneath 10wt.% NF-M, NF-H drives the transition to the disordered phase, and above which NF-H increases interfilament spacing. GENERAL SIGNIFICANCE Understanding the role of individual subunits in regulating the network structure will enable us to understand the mechanisms that drive the dysfunction of these networks, as observed in diseased conditions.

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 αβ-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.

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.

Osmotic Shock-Triggered Assembly of Highly Charged, Nanoparticle-Supported Membranes

Spherical nanoparticle-supported lipid bilayers (SSLBs) combine precision nanoparticle engineering with biocompatible interfaces for various applications, ranging from drug delivery platforms to structural probes for membrane proteins. Although the bulk, spontaneous assembly of vesicles and larger silica nanoparticles (>100 nm) robustly yields SSLBs, it will only occur with low charge density vesicles for smaller nanoparticles (<100 nm), a fundamental barrier in increasing SSLB utility and efficacy. Here, through whole mount and cryogenic transmission electron microscopy, we demonstrate that mixing osmotically loaded vesicles with smaller nanoparticles robustly drives the formation of SSLBs with high membrane charge density (up to 60% anionic lipid or 50% cationic lipid). We show that the osmolyte load necessary for SSLB formation is primarily a function of absolute membrane charge density and is not lipid headgroup-dependent, providing a generalizable, tunable approach toward bulk production of highly curved and charged SSLBs with various membrane compositions.

Mapping the Interactions of Alpha-Synuclein to Lipid Membranes in the Physiological Limit

protein-membrane interactions. Making use of recently developed protocol and assays for the preparation and characterization of asymmetric lipid vesicles, we studied compositionally and isotopically asymmetric proteoliposomes containing gramicidin. Protein incorporation, conformation and function were examined with small-angle x-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay. Differential scanning calorimetry revealed the effect of the protein on the melting transition temperatures of the two bilayer leaflets, which over time merged into a single peak indicating lipid scrambling. Using proton NMR, we monitored the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles with and without the protein. Our results show that gramicidin increases lipid flip-flop in a concentrationdependent manner.

Expression and isolation of recombinant tau

In this chapter, we describe methods for the purification of both untagged and polyhistidine-tagged tau protein. These protocols utilize a bacterial expression system to produce the tau isoform of interest, followed by heat treatment and column chromatography to separate tau from impurities. These techniques yield a biochemically pure protein with which to pursue any number of questions regarding the mechanisms of tau action.

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.

The Effect of Multivalent Cations on Microtubule-Protein Tau Ordering

In previous studies, it was found that paclitaxel-stabilized microtubules (MTs), hollow protein nanotubes comprised of assembled αβ-tubulin heterodimers, spontaneously assemble into bundles above a critical concentration (∼1.5 mM Spermine) of tetravalent spermine (PNAS 2004, 101, 16099). Further, at concentrations of spermine several-fold higher (∼10 mM Spermine), paclitaxel-stabilized MT bundles (BMT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (BITT), the outside surface of which corresponds to the inner surface of the BMT tubules (Nature Materials 2014, 13, 195). Here we will report on our current study of protein Tau-microtubule ordering (in an active system) in the absence of paclitaxel and, in particular, the effect of biological cations on microtubule self-assembly using transmission electron microscopy (TEM) and synchrotron small-angle X-ray scattering (SAXS).

Responsive and Tunable Neurofilament Protein Hydrogel Assemblies - A Synchrotron X-Ray Scattering Study of Composition and Salt Dependent Response

Neurofilaments (NFs) are the intermediate filaments in neuronal cells that along with other cytoskeletal network structures play a major role in the mechanical integrity of neuronal processes. NFs are assembled from three different subunits (NF-Low (NF-L), NF-Medium (NF-M), NF-High (NF-H)) that differ mainly in the sequence length of their unstructured C-terminal sidearms. The sidearms direct the lateral associations between filaments thus forming the NF hydrogel networks (the physiologically relevant NF protein assembly state in the axoplasm). The interfilament lateral associations are predominantly electrostatic, enabled by the polyampholytic nature of the sidearms. We examine their strength and range by varying the salinity of the in vitro buffer. Furthermore, motivated by variable in vivo subunit expression in axons versus dendrites that results in variable network packing, reassembled (in vitro) binary system NF-hydrogels have revealed the different contributions of individual subunits to interfilament interactions and to network interfilament spacings [1]. Synchrotron x-ray scattering experiments have allowed us to study the changes in the microscopic structure of NF hydrogels as a function of salt and sidearm density. At high weight ratios of NF-M and NF-H, and as a function of increasing salt concentrations, NF gels exhibit an unexpectedly abrupt transition from a weakly oriented (nearly isotropic) low filament density gel with interfilament spacing d=1000A to a highly oriented liquid crystalline gel with high filament density and d=500A (NF-M) and 700A (NF-H). The tunability of the network in vitro mirrors in vivo cellular control of the NF network via subunit phosphorylation, which may transition the network from a highly oriented rigid state to one with orientational plasticity.Funded by DOE-BES-DE-FG-02-06ER46314 and NSF-DMR-1101900[1] R. Beck, J. Deek, J.B. Jones, C.R. Safinya. Nature Materials. 9, 40 (2010).