This review is part of a special memorial issue in honor of Erich Sackmann's remarkable research career, in particular on the biophysics of equilibrium and living membranes. Erich's large body of discoveries firmly established him as a giant in the field of biophysics. In our review, we describe structural studies of microtubules (MTs) in the presence of the microtubule-associated protein tau, an intrinsically disordered protein confined to the axon of mature neurons in vertebrates. We start with a brief review of experiments where the inherently dynamical MT is fixed with the cancer chemotherapy drug molecule paclitaxel. At a paclitaxel/tubulin-dimer molar ratio of 1, tau is found to modulate the protofilament number of MTs with a tendency to increase the MT diameter. Under these high paclitaxel/tubulin molar ratios, where a vast amount of comprehensive literature on the high-resolution structure of tubulin and MTs exists, tau does not mediate bundling interactions between MTs. The review further describes more recent structural studies, which combine synchrotron small-angle x-ray scattering (SAXS) with electron microscopy of plastic-embedded preparations. The studies focus on elucidating the steady-state assembled structures emerging from reaction mixtures of tubulin, tau, and GTP at 37°C, all in the "absence" of paclitaxel. The mixtures contained either 1 millimolar (mM) Mg2+ present in the buffer or up to an additional few mM of Mg2+ comparable to cellular concentrations. The latter mixtures are intended to provide samples for studies to clarify the effects of cellular metal ions on tubulin/tau assemblies. The combined paclitaxel-free studies represent a minimal cell-free model of the MT cytoskeleton found in the central core of the axon initial segment of mature neurons. SAXS and TEM revealed out-of-equilibrium steady-state structures with hexagonal and linear symmetries (in cross section) and large MT wall-to-wall spacing greater than the diameter of MTs. The spacings and "linear MT architecture" resemble the morphology of MT fascicles found in the axon initial segment. SAXS of reaction mixtures combined with TEM imaging of plastic-embedded samples provided evidence that microtubules are bundled and stabilized by an "intervening network" of complexes of tubulin oligomers and tau.

Microtubules (MTs) are a major component of the eukaryotic cytoskeleton. MT architecture is highly regulated by MT-associated proteins such as Tau as well as a number of MT-targeted chemotherapeutic agents such as paclitaxel (PTX). In this study, we examined the ability of each of the six different alternatively spliced isoforms of human wild-type (WT) Tau (4R2N, 4R1N, 4R0N, 3R2N, 3R1N, and 3R0N) and PTX to bind to MTs as well as their effects upon MT structure. MTs were assembled in the physiologically relevant experimental regime of mixing WT Tau protein with unpolymerized tubulin and then treating the resulting MTs with PTX (i.e., Tau-coassembled MTs). The extent of Tau and PTX binding to MTs was assayed by co-sedimentation/Western blotting and high-performance liquid chromatography, respectively. Radial size of MTs was determined by synchrotron small-angle x-ray scattering. We observed that 4R Tau and PTX compete for binding to MTs, whereas 3R Tau and PTX exhibit only limited competition. These observations suggest that both 4R and 3R Tau bind initially to the well-studied binding sites on the outer surface of MTs, followed by binding to the less-well-understood binding site within the MT lumen in an isoform-specific manner. These binding events also lead to distinct effects on MT radial structure compared with MTs formed by PTX and then treated with Tau (i.e., PTX-stabilized MTs). Specifically, the inner radius of MTs first increased and then markedly decreased with increasing Tau concentrations. In addition to providing fundamental insights in the basic biochemistry of MTs, our results have implications regarding the onset and progression of chemotherapy-induced peripheral neuropathy, a consequence of many MT-targeted anticancer therapeutics including PTX. The differential use of the luminal Tau binding site in 4R versus 3R further raises the possibility of differential Tau isoform action in fetal versus adult nervous systems.

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 Mg 2+ or Ca 2+ divalent cations in mixtures containing αβ-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 (B ws ), a transient intermediate MT bundle phase (B int ), 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, αβ-tubulin oligomers in the IN are crosslinked by tau’s MT binding repeats, which also link αβ-tubulin oligomers to αβ-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.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein that binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full-length human protein tau and other negatively charged binding substrates, as revealed by differential interference contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase-separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes (i.e. tau-lipoplexes) exhibited distinct types of morphologies. This included large ∼20-30 μm tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles and tau-mediated finite-size assemblies of small liposomes. As the salt concentration was increased to near and above 150 mM for 1:1 electrolytes, AL-tau complexes remained stable, while tau self-coacervate droplets were found to dissolve, indicative of the breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen-facing monolayer of the axon's plasma membrane, suggesting the possibility of transient yet robust interactions near relevant ionic strengths found in neurons.

Aggregated and hyperphosphorylated Tau is one of the pathological hallmarks of Alzheimer’s disease. Tau is a polyampholytic and intrinsically disordered protein (IDP). In this paper, we present for the first time experimental results on the ionic strength dependence of the radius of gyration (Rg) of human Tau 4RS and 4RL isoforms. Synchrotron X-ray scattering revealed that 4RS Rg is regulated from 65.4 to 58.5 Å and 4RL Rg is regulated from 70.9 to 57.9 Å by varying ionic strength from 0.01 to 0.592 M. The Rg of 4RL Tau is larger than 4RS at lower ionic strength. This result provides an insight into the ion-responsive nature of intrinsically disordered and polyampholytic Tau, and can be implicated to the further study of Tau-Tau and Tau-tubulin intermolecular structure in ionic environments.

Tau is an intrinsically disordered neuronal protein known to modulate microtubule dynamics through the suppression of dynamic instability. Recent studies have shown that Tau mediates dynamical linear microtubule bundles similar to microtubule fascicles observed in vivo, which are a cardinal feature of the axon initial segment (P. J. Chung, C. Song, et al. Nature Communications 2016, 7, 12278. DOI: 10.1038/ncomms12278 and ACS Macro Lett. 2018, 7, 228-232. DOI: 10.1021/acsmacrolett.7b00937). In order to better understand tau's role in the physiologic bundling of MTs, quantitative techniques of synchrotron small-angle x-ray scattering data—combined with plastic-embedded TEM, and other analytical techniques—have been utilized. Here we show that Ca2+ and Mg2+ in the mM concentration range (corresponding to the average concentration in cells for Mg2+) can destabilize steady-state, bundled microtubules (in the presence of GTP at 37 °C), inducing a novel bundle-to-bundle transition prior to the tau-coated tubulin ring state. This transitory bundled phase suggests that tubulin oligomers also play a role in stabilizing MT bundles.

Supplementary Figure 1, Table 1 from Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of β-tubulin at Cys 12 and suppression of microtubule dynamics

File contains description of supplementary methods used for sciatic nerve immunofluorescence data generation and quantification with Supplementary Fig 1 showing how raw fluorescence intensity values were normalized in order to compare levels of acetylated alpha tubulin from samples across treatment groups. Supplementary Figs 2 thru 4 show DRG and sciatic nerve morphology/morphometrics for chemotherapies other than PACLI. Supplementary Table 1 adds perspective to how MTD used to compare the chemotherapies in this study relates to their pharmacokinetic and IC50 properties.

The emergence of the SARS-CoV-2 Omicron variant in 2021 is associated with a global surge of cases in late 2021 and early 2022. Identifying the introduction of novel SARS-CoV-2 variants to a population is imperative to inform decisions by clinicians and public health officials. Here, we describe a quantitative reverse transcription PCR-based assay (RT-qPCR) targeting unique mutations in the Omicron BA.1/BA1.1 and BA.2 viral genomes. This assay accurately and precisely detect the presence of these Omicron variants in patient samples in less than four hours. Using this assay, we tested 270 clinical samples and detected the introduction of Omicron BA.1/BA1.1 and BA.2 in the Santa Barbara County (SBC) population in December 2021 and February 2022, respectively. Identifying Omicron variants using this RT-qPCR assay showed complete concordance with whole viral genome sequencing; both assays indicated that Omicron was the dominant variant in SB County. Our data substantiate that RT-qPCR-based virus detection assays offer a fast and inexpensive alternative to NGS for virus variant-specific detection approach, which allows streamlining the detection of Omicron variants in patient samples.

Tau, is an important microtubule-associated protein (MAP) essential in regulating microtubule (MT) dynamics in neuronal axons. A central role of tau is the stabilization of MTs through the suppression of dynamic instability in a tau-concentration dependent manner. Tau-MT associations are made possible through Tau’s MT binding domains and non-specific electrostatic interactions driven in part by the polyampholytic nature of tau and the lack of a stable tertiary structure. In fact it is known that tau is a surface active molecule that, upon chemical alterations, can interact pathologically with itself to promote neurodegenerative tauopathies such as Alzheimer’s and frontotemporal dementia with parkinsonism-17 (FTDP-17).

Protein tau and isoforms are essential microtubule regulating proteins found in the axons of healthy mature neurons. Tau is an intrinsically disordered protein and a polyampholyte. One central role of tau is in regulation of microtubule dynamic instability in neurons. However, tau's flexibility and gradient charge distribution increases the propensity for inter-molecular interactions with other charged species in the cytosol. In fact it is known that tau is a surface active molecule that, upon chemical alterations, can interact pathologically with itself to promote neurodegenerative states. While there is incomplete understanding of the mechanisms driving neurodegenerative pathology, some evidence exists that tau can react with anionic macromolecules within the cytosol. For instance, in the pathogenesis of Alzheimer's disease, it has been postulated that anionic lipid membranes may play a role in tau permeabilization leading to disease progression. However, the extent and conditions of these interactions are still poorly understood. Our work is centered on understanding the structural implications of the interactions of full length (4RL) tau with anionic biological lipid membranes. To understand the nature of the electrostatic interactions in tau/anionic liposome mixtures, we probed phase transitions of fluorescently tagged 4RL tau and anionic liposomes: A.) in biologically relevant salt concentrations and B.) at different lipid/tau charge ratios (ρ<1, ρ=1, and ρ>1). We showcase our findings using differential interference contrast microscopy (DIC) and fluorescence microscopy.

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.

The microtubule-associated protein tau is known for its ability to alter microtubule (MT) dynamic instability in developing and mature neurons and for its role in the pathology of many neurodegenerative diseases. Bundling of Mts into small linear arrays (or fascicles) is a cardinal feature of the axon initial segment. While tau plays an important role in the structural stability of MT fascicles, the exact mechanism by which tau imparts MT bundle stabilization remains unclear. To help elucidate the nature of tau's role in physiological bundling of Mts, we have expanded on a recent platform for studying paclitaxel-free in vitro reaction mixtures of tubulin, GTP, and wild-type tau under dissipative, out-of-equilibrium conditions at 37 °C.

The microtubule inhibitor (MTI) class of chemotherapeutics provide an effective treatment for several different types of cancers, however, severe chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting toxicity in patients that limits their use. While CIPN was predicted with MTIs based on histopathology and functional effects in non-clinical toxicology studies, these investigations often require large numbers of animals and long term studies. As in vitro MT assays have been used for decades to study mechanisms of efficacy, we hypothesized that those same assays could be used to study mechanisms of peripheral neuropathy and predict severe CIPN. We analyzed published data on in vitro microtubule (MT) properties for different MTIs that cause varying levels of peripheral neuropathy in patients. Eribulin, vinorelbine and vinfluinine, which all have less severe CIPN than the vinca alkaloids or taxanes, have unique MT properties consisting of reduced affinity and limited binding to MTs (i.e. bind only to the ends and not along the length). Binding more potently to tubulin in the absence of neuronal Bill tubulin was also observed with eribulin and may suggest specificity for tumor tubulin over neuronal tubulin. These are possible mechanisms for causing less severe deleterious effects on MTs in peripheral nerves leading to reduced severity of CIPN. Our analyses demonstrated that in vitro tools used to study the mechanisms of action in inducing severe CIPN (i.e MTI interactions with MTs) warrant further investigation and may be useful for developing next generation MTIs with reduced CIPN.

Microtubules are dynamic, hierarchically assembled protein structures whose various functional abilities are strongly tied to their ability to switch between periods of slow polymerization and rapid depolymerization. In the axons of neurons, microtubule function is also dependent on collective phase behavior, where, for example, homogenously distributed microtubules along the axon body act as cytoskeletal support for axoplasmic transport. In contrast, high-density, phase-separated bundles of microtubules act at the axon initial segment as diffusion barriers and in axonal protrusions to sustain elongation and branching. While dynamic instability is tightly regulated by function- and cell-specific microtubule-associated proteins (MAPs), the axon-specific neuronal MAP tau is thought to both suppress dynamic instability in the body of the axon and to promote bundling of microtubules in the peripheral regions (proximal and distal). Accordingly, in vitro assays have confirmed tau's role in these two distinct microtubule properties (dynamic instability and bundle formation), but the role of GTP and Mg2+ (necessary for tubulin polymerization in vitro and in vivo) in modulating tau-mediated microtubule-microtubule interactions is poorly understood. In many experiments, reaction mixtures tend to contain both molecules in excess to ensure polymerization, but recent results from our group show that tau's ability to regulate both bundling and polymerization is strongly dependent on the relative concentrations of both GTP and Mg2+ and follows unexpected trends.

Tubulins heterodimers are pre-programmed biological building blocks. αβ-tubulins are assembled into cytoskeleton microtubules (MTs), which are dynamic protein nanotubes involved in many important cellular functions. During the growth and shrinkage of MTs, the conformational changes of tubulin building blocks occur upon the hydrolysis of nucleotide bound to tubulin. The tubulin assembly is known to be sensitive to various molecules such as MT-associated proteins (ex. Tau), drugs (ex. Taxol), and even cationic molecules (ex. divalent magnesium ions). We show our recent findings on the tubulin architectures in the presence of cationic polymers, in results which play the role as molecular switches. Structures of the assemblies were studied by using synchrotron small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM).

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.

Tau is a highly soluble intrinsically disordered protein (IDP) characterized classically as a microtubule-associated protein (MAP) in axons of mature neurons. The most well established primary function of tau is to suppress microtubule (MT) dynamic instability providing a stable foundation for organelle trafficking and cell communication. However, the dynamic and polyampholytic nature of tau's peptide composition implies a capacity to interact with charged biological substrates in the cytosol for other distinct functions. There is emerging evidence suggesting that tau associations with charged, specifically anionic, cytosolic molecules may prime pathological structural states seen in neurodegenerative diseases. However, the mechanisms of these secondary interactions are still poorly understood. Therefore, our goal is to understand the structural implications of tau associations with anionic molecules, specifically biological lipid membranes. Here we probe phase transitions of tau-anionic liposomes interactions at different anionic liposome formulations using differential interference contrast microscopy (DIC), fluorescence microscopy, and transmission electron microscopy (TEM).

Tau is an intrinsically disordered neuronal protein known to modulate microtubule dynamics through the suppression of dynamic instability. Recent studies have shown that Tau mediates dynamical linear microtubule bundles similar to microtubule fascicles observed in vivo, which are a cardinal feature of the axon initial segment (P. J. Chung, C. Song, et al. Nature Communications 2016, 7, 12278. DOI: 10.1038/ncomms12278 and ACS Macro Lett. 2018, 7, 228-232. DOI: 10.1021/acsmacrolett.7b00937). In order to better understand the architecture of these nanoscale energy-consuming dissipative structures, quantitative techniques of synchrotron small-angle x-ray scattering data—combined with TEM, and other analytical techniques—have been utilized. Here we show that under biologically relevant GTP and Mg2+ concentrations, microtubule bundles undergo a time induced transition, in which the wall to wall distance of the previously observed "pseudo-hexagonal" wide spacing dramatically drops, and remodels to form a tightly bound, "locked-hexagonal" structure, which in turn, induces a rapid depolymerization of the microtubule bundle. The structure and stability of these architectures appear to be Tau-isoform dependent and underscore the importance of the expression of distinct Tau-isoforms within neurons.