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.

Disorder of written expression and dysgraphia: definition, diagnosis, and management.

Writing is a complex task that is vital to learning and is usually acquired in the early years of life. 'Dysgraphia' and 'specific learning disorder in written expression' are terms used to describe those individuals who, despite exposure to adequate instruction, demonstrate writing ability discordant with their cognitive level and age. Dysgraphia can present with different symptoms at different ages. Different theories have been proposed regarding the mechanisms of dysgraphia. Dysgraphia is poorly understood and is often undiagnosed. It has a high rate of co-morbidity with other learning and psychiatric disorders. The diagnosis and treatment of dysgraphia and specific learning disorders typically centers around the educational system; however, the pediatrician can play an important role in surveillance and evaluation of co-morbidity as well as provision of guidance and support.

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.

α-Synuclein Sterically Stabilizes Spherical Nanoparticle-Supported Lipid Bilayers.

While it is generally accepted that neuronal protein α-synuclein binds to highly curved and highly charged lipid membranes, its biological function beyond binding remains unknown despite its fundamental link to Parkinson's disease. Herein, we utilize spherical nanoparticle lipid bilayers (SSLBs) to recapitulate the charge and curvature limit of membrane organelles with which α-synuclein associates and probe how α-synuclein affects SSLB structure and dynamics as a proxy for interorganelle interactions. Small-angle X-ray scattering and X-ray photon correlation spectroscopy reveal our SSLBs form aggregates that are clearly broken up by the addition of α-synuclein, a clear indication that α-synuclein confers steric stabilization to membrane surfaces.

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.

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.

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.

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.

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.

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.

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

Microtubules (MTs) are hollow cytoskeletal filaments assembled from αß-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.

Active Videogaming for Individuals with Severe Movement Disorders: Results from a Community Study.

OBJECTIVE: Active videogaming (AVG) has potential to provide positive health outcomes for individuals with cerebral palsy (CP), but their use for individuals with severe motor impairments is limited. Our objective was to evaluate the accessibility and enjoyment of videogames using the Kinect™ (Microsoft, Redmond, WA) with the Flexible Action and Articulated Skeleton Toolkit (FAAST) system (University of Southern California Institute for Creative Technologies, Los Angeles, CA) for individuals with severely limiting CP. MATERIALS AND METHODS: A videogaming system was installed in a community center serving adults with CP, and a staff member was instructed in its use. Participants completed a baseline survey assessing demographics, mobility, and prior videogame experience; they then used the FAAST system with Kinect and completed a 5-point Likert survey to assess their experience. Descriptive statistics assessed overall enjoyment of the system, and Mann-Whitney U tests were conducted to determine whether responses differed by demographic factors, mobility, or prior videogame experience. RESULTS: Twenty-two subjects were recruited. The enjoyment scale demonstrated high internal consistency (Cronbach's alpha=0.88). The mean total enjoyment score was 4.24 out of 5. Median scores did not significantly differ by ethnicity, gender, CP severity, or previous videogame exposure. CONCLUSIONS: The FAAST with Kinect is a low-cost system that engages individuals with severe movement disorders across a wide range of physical ability and videogame experience. Further research should be conducted on in-home use, therapeutic applications, and potential benefits for socialization.

Social Behaviors and Active Videogame Play in Children with Autism Spectrum Disorder.

OBJECTIVE: Children with autism spectrum disorder (ASD) often display problematic and excessive videogame play. Using active videogames (AVGs) may have physical benefits, but its effects on socialization are unknown. MATERIALS AND METHODS: We conducted an A-B-A' experiment comparing sedentary videogames and AVGs for three dyads of a child with ASD and his sibling. An augmented reality (AR) game was used to introduce AVGs. Sessions were coded for communication, positive affect, and aggression. RESULTS: One dyad had increases in positive affect with AVGs. Otherwise, social behaviors were unchanged or worse. The AR game demonstrated consistent elevations in social behaviors. CONCLUSIONS: Use of AVGs has inconsistent effects on social behavior for children with ASD. Further research is needed to understand mediators of response to AVGs. AR games should be evaluated for potential benefits on socialization and positive affect.

Neurofilament sidearms modulate parallel and crossed-filament orientations inducing nematic to isotropic and re-entrant birefringent hydrogels.

Neurofilaments are intermediate filaments assembled from the subunits neurofilament-low, neurofilament-medium and neurofilament-high. In axons, parallel neurofilaments form a nematic liquid-crystal hydrogel with network structure arising from interactions between the neurofilaments' C-terminal sidearms. Here we report, using small-angle X-ray-scattering, polarized-microscopy and rheometry, that with decreasing ionic strength, neurofilament-low-high, neurofilament-low-medium and neurofilament-low-medium-high hydrogels transition from the nematic hydrogel to an isotropic hydrogel (with random, crossed-filament orientation) and to an unexpected new re-entrant liquid-crystal hydrogel with parallel filaments--the bluish-opaque hydrogel--with notable mechanical and water retention properties reminiscent of crosslinked hydrogels. Significantly, the isotropic gel phase stability is sidearm-dependent: neurofilament-low-high hydrogels exhibit a wide ionic strength range, neurofilament-low-medium hydrogels a narrow ionic strength range, whereas neurofilament-low hydrogels lack the isotropic gel phase. This suggests a dominant regulatory role for neurofilament-high sidearms in filament reorientation plasticity, facilitating organelle transport in axons. Neurofilament-inspired biomimetic hydrogels should therefore exhibit remarkable structure-dependent moduli and slow and fast water-release properties.

Continuity of care in fixed-day versus variable-day resident continuity clinics.

OBJECTIVE: Since the introduction of resident work-hour standards, pediatric residency programs have struggled to preserve robust continuity clinic experiences. Many programs have resorted to more flexible approaches to resident scheduling. We know little regarding the impact of such changes. We compared 2 continuity clinic scheduling models: a traditional fixed-day clinic and a variable-day clinic in which resident clinic days vary each week to accommodate resident schedules. METHODS: The setting for our study was a large university resident continuity clinic. We analyzed 111 resident schedules and 1113 visits by children aged younger than 1 year during 2 periods: July 2007 to December 2007, when residents were scheduled by using a variable-day clinic model, and July 2008 to December 2008, when a fixed-day model was used. We compared the number of clinic sessions per resident and continuity of care. We used the usual provider of care definition of continuity: the proportion of visits in which a patient is seen by his or her primary resident. A multivariable logistic regression was used to model the relationship between patient continuity of care and clinic structure (fixed-day vs variable-day), resident level, patient age, and appointment type. RESULTS: The number of clinics per resident during a 6-month period was higher using variable-day scheduling (19.6 vs 16.2; P < .01), whereas continuity of care was lower (0.54 vs 0.61; P = .01) In the multivariate model, continuity of care was significantly higher under the fixed-day model (odds ratio 1.40; P < .01). CONCLUSIONS: Scheduling residents for continuity clinic on variable days results in lower patient continuity of care despite increased resident time in clinic.