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-lowhigh, 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 neurofilamentlow 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.

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.

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; Φ Tau = 1/ 10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to P B ∼ 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 (P B ∼ 1,000-2,000 Pa) over the same range. Remarkably, the abrupt increase in P B observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < Φ Tau < 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.Tau | intrinsically disordered proteins | microtubule | SAXS | force measurement

No abstract

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.

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.

Alpha-synuclein is a presynaptic protein linked to Parkinson’s disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1.

Investigating MAP-mediated interactions between microtubules (as it relates to in vivo behavior) requires the elimination or minimization of paclitaxel.