Poly(ferrocenylsilane)-Based Redox-Active Artificial Organelles for Biomimetic Cascade Reactions.

Artificial organelles serve as functional counterparts to natural organelles, which are primarily employed to artificially replicate, restore, or enhance cellular functions. While most artificial organelles exhibit basic functions, we diverge from this norm by utilizing poly(ferrocenylmethylethylthiocarboxypropylsilane) microcapsules (PFC MCs) to construct multifunctional artificial organelles through water/oil interfacial self-assembly. Within these PFC MCs, enzymatic cascades are induced through active molecular exchange across the membrane to mimic the functions of enzymes in mitochondria. We harness the inherent redox properties of the PFC polymer, which forms the membrane, to facilitate in-situ redox reactions similar to those supported by the inner membrane of natural mitochondria. Subsequent studies have demonstrated the interaction between PFC MCs and living cell including extended lifespans within various cell types. We anticipate that functional PFC MCs have the potential to serve as innovative platforms for organelle mimics capable of executing specific cellular functions.

Effect of ionic strength on the assembly of simian vacuolating virus capsid protein around poly(styrene sulfonate).

Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent, such as RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with a combinatorial number of intermediates. Hence, the mechanism of the reaction is poorly understood. In this paper, we combined solution small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (TEM), and computational modeling to determine the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by SAXS and cryo-TEM. To analyze the data, we performed Langevin dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures consistent with the experimental data. We found that close to physiological ionic strength, [Formula: see text] VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and [Formula: see text] particles. Including the simulated structures was essential to explain the SAXS data in a manner that is consistent with the cryo-TEM images.

Sound-mediated nucleation and growth of amyloid fibrils.

Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, influencing their stability, folding patterns, and self-assembling behavior. Regarding understanding the effects of ultrasound on the self-assembly of amyloidogenic proteins, our knowledge remains quite limited. In our recent work, we established the boundary conditions under which sound energy can either cause damage or induce only negligible changes in the structure of protein species. In the present study, we demonstrate that when the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, as it has been revealed by MD, which is sufficient for primary nucleation, characterized by adopting a hydrogen-bonded ß -sheet-rich structure. These structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from monomeric lysozyme proteins, in a controlled manner, until they reach a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed under the influence of ultrasound energy and natively fibrillated lysozyme, share identical structural characteristics. Thus, these results contribute to our understanding of the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential exploitation of sound energy in a protein chemistry environment.

Insight into structural biophysics from solution X-ray scattering.

The current challenges of structural biophysics include determining the structure of large self-assembled complexes, resolving the structure of ensembles of complex structures and their mass fraction, and unraveling the dynamic pathways and mechanisms leading to the formation of complex structures from their subunits. Modern synchrotron solution X-ray scattering data enable simultaneous high-spatial and high-temporal structural data required to address the current challenges of structural biophysics. These data are complementary to crystallography, NMR, and cryo-TEM data. However, the analysis of solution scattering data is challenging; hence many different analysis tools, listed in the SAS Portal (http://smallangle.org/), were developed. In this review, we start by briefly summarizing classical X-ray scattering analyses providing insight into fundamental structural and interaction parameters. We then describe recent developments, integrating simulations, theory, and advanced X-ray scattering modeling, providing unique insights into the structure, energetics, and dynamics of self-assembled complexes. The structural information is essential for understanding the underlying physical chemistry principles leading to self-assembled supramolecular architectures and computational structural refinement.

Upgrade of D+ software for hierarchical modeling of X-ray scattering data from complex structures in solution, fibers and single orientations.

This article presents an upgrade of the D+ software [Ginsburg et al. (2019 ▸). J. Appl. Cryst. 52, 219-242], expanding its hierarchical solution X-ray scattering modeling capabilities for fiber diffraction and single crystallographic orientations. This upgrade was carried out using the reciprocal grid algorithm [Ginsburg et al. (2016 ▸). J. Chem. Inf. Model. 56, 1518-1527], providing D+ its computational strength. Furthermore, the extensive modifications made to the Python API of D+ are described, broadening the X-ray analysis performed with D+ to account for the effects of the instrument-resolution function and polydispersity. In addition, structure-factor and radial-distribution-function modules were added, taking into account the effects of thermal fluctuations and intermolecular interactions. Finally, numerical examples demonstrate the usage and potential of the added features.

Modulating amyloids' formation path with sound energy.

Protein folding is crucial for biological activity. Proteins' failure to fold correctly underlies various pathological processes, including amyloidosis, the aggregation of insoluble proteins (e.g., lysozymes) in organs. The exact conditions that trigger the structural transition of amyloids into ß-sheet-rich aggregates are poorly understood, as is the case for the amyloidogenic self-assembly pathway. Ultrasound is routinely used to destabilize a protein's structure and enhance amyloid growth. Here, we report on an unexpected ultrasound effect on lysozyme amyloid species at different stages of aggregation: ultrasound-induced structural perturbation gives rise to nonamyloidogenic folds. Our infrared and X-ray analyses of the chemical, mechanical, and thermal effects of sound on lysozyme's structure found, in addition to the expected ultrasound-induced damage, evidence of irreversible disruption of the ß-sheet fold of fibrillar lysozyme resulting in their structural transformation into monomers with no ß-sheets. This structural transition is reflected in changes in the kinetics of protein self-assembly, namely, either prolonged nucleation or accelerated fibril growth. Using solution X-ray scattering, we determined the structure, the mass fraction of lysozyme monomer, and the morphology of its filamentous assemblies formed under different sound parameters. A nanomechanical analysis of ultrasound-modified protein assemblies revealed a correlation between the ß-sheet content and elastic modulus of the protein material. Suppressing one of the ultrasound-derived effects allowed us to control the structural transformations of lysozyme. Overall, our comprehensive investigation establishes the boundary conditions under which ultrasound damages protein structure and fold. This knowledge can be utilized to impose medically desirable structural modifications on amyloid ß-sheet-rich proteins.

Effect of tubulin self-association on GTP hydrolysis and nucleotide exchange reactions.

We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. Both reactions are relevant for microtubule (MT) dynamics. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied by determining the concentrations of tubulin-free and tubulin-bound GTP and GDP. We observed no GTP hydrolysis below the critical conditions for MT assembly (either below the critical tubulin concentration and/or at low temperature), despite the assembly of tubulin 1D curved oligomers and single-rings, showing that their assembly did not involve GTP hydrolysis. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Cryo-TEM images showed that GTP-tubulin 1D oligomers were curved also at 36 °C. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to determine the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites. This conclusion may have implications on the dynamics at the tip of the MT plus end.

Dehydration does not affect lipid-based hydration lubrication.

Phosphatidylcholine (PC) lipid bilayers at surfaces massively reduce sliding friction, via the hydration lubrication mechanism acting at their highly-hydrated phosphocholine headgroups, a central paradigm of biological lubrication, particularly at articular cartilage surfaces where low friction is crucial for joint well-being. Nanotribological measurements probed the effect on such lubrication of dehydration by dimethyl sulfoxide (DMSO), known to strongly dehydrate the phosphocholine headgroups of such PC bilayers, i.e. reduce the thickness of the inter-bilayer water layer, and thus expected to substantially degrade the hydration lubrication. Remarkably, and unexpectedly, we found that the dehydration has little effect on the friction. We used several approaches, including atomic force microscopy, small- and wide-angle X-ray scattering and all-atom molecular dynamics simulations to elucidate this. Our results show that while DMSO clearly removes hydration water from the lipid head-groups, this is offset by both higher areal head-group density and by rigidity-enhancement of the lipid bilayers, both of which act to reduce frictional dissipation. This sheds strong light on the robustness of lipid-based hydration lubrication in biological systems, despite the ubiquitous presence of bio-osmolytes which compete for hydration water.

Mechanism of the Initial Tubulin Nucleation Phase.

Tubulin nucleation is a highly frequent event in microtubule (MT) dynamics but is poorly understood. In this work, we characterized the structural changes during the initial nucleation phase of dynamic tubulin. Using size-exclusion chromatography-eluted tubulin dimers in an assembly buffer solution free of glycerol and tubulin aggregates enabled us to start from a well-defined initial thermodynamic ensemble of isolated dynamic tubulin dimers and short oligomers. Following a temperature increase, time-resolved X-ray scattering and cryo-transmission electron microscopy during the initial nucleation phase revealed an isodesmic assembly mechanism of one-dimensional (1D) tubulin oligomers (where dimers were added and/or removed one at a time), leading to sufficiently stable two-dimensional (2D) dynamic nanostructures, required for MT assembly. A substantial amount of tubulin octamers accumulated before two-dimensional lattices appeared. Under subcritical assembly conditions, we observed a slower isodesmic assembly mechanism, but the concentration of 1D oligomers was insufficient to form the multistranded 2D nucleus required for MT formation.

Mechanism of Tubulin Oligomers and Single-Ring Disassembly Catastrophe.

Cold tubulin dimers coexist with tubulin oligomers and single rings. These structures are involved in microtubule assembly; however, their dynamics are poorly understood. Using state-of-the-art solution synchrotron time-resolved small-angle X-ray scattering, we discovered a disassembly catastrophe (half-life of ∼0.1 s) of tubulin rings and oligomers upon dilution or addition of guanosine triphosphate. A slower disassembly (half-life of ∼38 s) was observed following an increase in temperature. Our analysis showed that the assembly and disassembly processes were consistent with an isodesmic mechanism, involving a sequence of reversible reactions in which dimers were rapidly added or removed one at a time, terminated by a 2 order-of-magnitude slower ring-closing/opening step. We revealed how assembly conditions varied the mass fraction of tubulin in each of the coexisting structures, the rate constants, and the standard Helmholtz free energies for closing a ring and for longitudinal dimer-dimer associations.

Structure and Energetics of GTP- and GDP-Tubulin Isodesmic Self-Association.

Tubulin self-association is a critical process in microtubule dynamics. The early intermediate structures, energetics, and their regulation by fluxes of chemical energy, associated with guanosine triphosphate (GTP) hydrolysis, are poorly understood. We reconstituted an in vitro minimal model system, mimicking the key elements of the nontemplated tubulin assembly. To resolve the distribution of GTP- and guanosine diphosphate (GDP)-tubulin structures, at low temperatures (∼10 °C) and below the critical concentration for the microtubule assembly, we analyzed in-line size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS) chromatograms of GTP- and GDP-tubulin solutions. Both solutions rapidly attained steady state. The SEC-SAXS data were consistent with an isodesmic thermodynamic model of longitudinal tubulin self-association into 1D oligomers, terminated by the formation of tubulin single rings. The analysis showed that free dimers coexisted with tetramers and hexamers. Tubulin monomers and lateral association between dimers were not detected. The dimer-dimer longitudinal self-association standard Helmholtz free energies were -14.2 ± 0.4 kBT (-8.0 ± 0.2 kcal mol-1) and -13.1 ± 0.5 kBT (-7.4 ± 0.3 kcal mol-1) for GDP- and GTP-tubulin, respectively. We then determined the mass fractions of dimers, tetramers, and hexamers as a function of the total tubulin concentration. A small fraction of stable tubulin single rings, with a radius of 19.2 ± 0.2 nm, was detected in the GDP-tubulin solution. In the GTP-tubulin solution, this fraction was significantly lower. Cryo-TEM images and SEC-multiangle light-scattering analysis corroborated these findings. Our analyses provide an accurate structure-stability description of cold tubulin solutions.

Hierarchical Assembly Pathways of Spermine-Induced Tubulin Conical-Spiral Architectures.

Tubulin, an essential cytoskeletal protein, assembles into various morphologies by interacting with an array of cellular factors. One of these factors is the endogenous polyamine spermine, which may promote and stabilize tubulin assemblies. Nevertheless, the assembled structures and their formation pathways are poorly known. Here we show that spermine induced the in vitro assembly of tubulin into several hierarchical architectures based on a tubulin conical-spiral subunit. Using solution X-ray scattering and cryo-TEM, we found that with progressive increase of spermine concentration tubulin dimers assembled into conical-frustum-spirals of increasing length, containing up to three helical turns. The subunits with three helical turns were then assembled into tubules through base-to-top packing and formed antiparallel bundles of tubulin conical-spiral tubules in a distorted hexagonal symmetry. Further increase of the spermine concentration led to inverted tubulin tubules assembled in hexagonal bundles. Time-resolved experiments revealed that tubulin assemblies formed at higher spermine concentrations assembled from intermediates, similar to those formed at low spermine concentrations. These results are distinct from the classical transition between twisted ribbons, helical, and tubular assemblies, and provide insight into the versatile morphologies that tubulin can form. Furthermore, they may contribute to our understanding of the interactions that control the composition and construction of protein-based biomaterials.

Effect of the ammonium salt anion on the structure of doxorubicin complex and PEGylated liposomal doxorubicin nanodrugs.

BACKGROUND: In Doxil®, PEGylated nanoliposomes are created by hydration of the lipids in ammonium sulfate, and are remotely loaded with doxorubicin by a transmembrane ammonium gradient. The ammonium sulfate is then removed from the external aqueous phase, surrounding the liposomes, and replaced by an isoosmotic sucrose solution in 10 mM histidine buffer at pH 6.5. METHODS: We prepared PEGylated liposomal doxorubicin (PLD) with a series of ammonium monovalent salts that after remote loading became the intraliposome doxorubicin counteranions. We analyzed the liposomes by solution X-ray scattering, differential scanning calorimetry, and electron micropscopy. RESULTS: PLDs prepared with sulfonic acid derivatives as counteranion exhibited chemical and physical stabilities. We determined the effect of these ammonium salt counteranions on the structure, morphology, and thermotropic behavior of the PEGylated nanoliposomes, formed before and after doxorubicin loading, and the bulk properties of the doxorubicin-counteranion complexes. By comparing the structure of the doxorubicin complexes in the bulk and inside the nanoliposomes, we revealed the effect of confinement on the structure and doxorubicin release rate for each of the derivatives of the ammonium sulfonic acid counteranions. CONCLUSIONS: We found that the extent and direction of the doxorubicin confinement effect and its release rate were strongly dependent on the type of counteranion. The counteranions, however, neither affected the structure and thermotropic behavior of the liposome membrane, nor the thickness and density of the liposome PEG layers. In an additional study, it was demonstrated that PLD made with ammonium-methane sulfonate exhibit a much lower Hand and Foot syndrome. GENERAL SIGNIFICANCE: The structure, physical state, and pharmacokinetics of doxorubicin in PEGylated nanoliposomes, prepared by transmembrane remote loading using gradients of ammonium salts, strongly depend on the counteranions.

Bridges of Calcium Bicarbonate Tightly Couple Dipolar Lipid Membranes.

The interaction between lipid membranes and ions is associated with a range of key physiological processes. Most earlier studies have focused on the interaction of lipids with cations, while the specific effects of the anions have been largely overlooked. Owing to dissolved atmospheric carbon dioxide, bicarbonate is an important ubiquitous anion in aqueous media. In this paper, we report on the effect of bicarbonate anions on the interactions between dipolar lipid membranes in the presence of previously adsorbed calcium cations. Using a combination of solution X-ray scattering, osmotic stress, and molecular dynamics simulations, we followed the interactions between 1,2-didodecanoyl-sn-glycero-3-phosphocholine (DLPC) lipid membranes that were dialyzed against CaCl2 solutions in the presence and absence of bicarbonate anions. Calcium cations adsorbed onto DLPC membranes, charge them, and lead to their swelling. In the presence of bicarbonate anions, however, the calcium cations can tightly couple one dipolar DLPC membrane to the other and form a highly condensed and dehydrated lamellar phase with a repeat distance of 3.45 ± 0.02 nm. Similar tight condensation and dehydration has only been observed between charged membranes in the presence of multivalent counterions. Bridging between bilayers by calcium bicarbonate complexes induced this arrangement. Furthermore, in this condensed phase, lipid molecules and adsorbed ions were arranged in a two-dimensional oblique lattice.

3D Printing of Ordered Mesoporous Silica Complex Structures.

Ordered mesoporous silica materials gain high interest because of their potential applications in catalysis, selective adsorption, separation, and controlled drug release. Due to their morphological characteristics, mainly the tunable, ordered nanometric pores, they can be utilized as supporting hosts for confined chemical reactions. Applications of these materials, however, are limited by structural design. Here, we present a new approach for the 3D printing of complex geometry silica objects with an ordered mesoporous structure by stereolithography. The process uses photocurable liquid compositions that contain a structure-directing agent, silica precursors, and elastomer-forming monomers that, after printing and calcination, form porous silica monoliths. The objects have extremely high surface area, 1900 m2/g, and very low density and are thermally and chemically stable. This work enables the formation of ordered porous objects having complex geometries that can be utilized in applications in both the industry and academia, overcoming the structural limitations associated with traditional processing methods.

Rapidly Forming Early Intermediate Structures Dictate the Pathway of Capsid Assembly.

There are ∼1030 possible intermediates on the assembly path from hepatitis B capsid protein dimers to the 120-dimer capsid. If every intermediate was tested, assembly would often get stuck in an entropic trap and essentially every capsid would follow a unique assembly path. Yet, capsids assemble rapidly with minimal trapped intermediates, a realization of the Levinthal paradox. To understand the fundamental mechanisms of capsid assembly, it is critical to resolve the early stages of the reaction. We have used time-resolved small angle X-ray scattering, which is sensitive to solute size and shape and has millisecond temporal resolution. Scattering curves were fit to a thermodynamically curated library of assembly intermediates, using the principle of maximum entropy. Maximum entropy also provides a physical rationale for the selection of species. We found that the capsid assembly pathway was exquisitely sensitive to initial assembly conditions. With the mildest conditions tested, the reaction appeared to be two-state from dimer to 120-dimer capsid with some dimers-of-dimers and trimers-of-dimers. In slightly more aggressive conditions, we observed transient accumulation of a decamer-of-dimers and the appearance of 90-dimer capsids. In conditions where there is measurable kinetic trapping, we found that highly diverse early intermediates accumulated within a fraction of a second and propagated into long-lived kinetically trapped states (≥90-mer). In all cases, intermediates between 35 and 90 subunits did not accumulate. These results are consistent with the presence of low barrier paths that connect early and late intermediates and direct the ultimate assembly path to late intermediates where assembly can be paused.

Assembly and Stability of Simian Virus 40 Polymorphs.

Understanding viral assembly pathways is of critical importance to biology, medicine, and nanotechology. Here, we study the assembly path of a system with various structures, the simian vacuolating virus 40 (SV40) polymorphs. We simulate the templated assembly process of VP1 pentamers, which are the constituents of SV40, into icosahedal shells made of N = 12 pentamers (T = 1). The simulations include connections formed between pentamers by C-terminal flexible lateral units, termed here "C-terminal ligands", which are shown to control assembly behavior and shell dynamics. The model also incorporates electrostatic attractions between the N-terminal peptide strands (ligands) and the negatively charged cargo, allowing for agreement with experiments of RNA templated assembly at various pH and ionic conditions. During viral assembly, pentamers bound to any template increase its effective size due to the length and flexibility of the C-terminal ligands, which can connect to other VP1 pentamers and recruit them to a partially completed capsid. All closed shells formed other than the T = 1 feature the ability to dynamically rearrange and are thus termed "pseudo-closed". The N = 13 shell can even spontaneously "self-correct" by losing a pentamer and become a T = 1 capsid when the template size fluctuates. Bound pentamers recruiting additional pentamers to dynamically rearranging capsids allow closed shells to continue growing via the pseudo-closed growth mechanism, for which experimental evidence already exists. Overall, we show that the C-terminal ligands control the dynamic assembly paths of SV40 polymorphs.

pH stability and disassembly mechanism of wild-type simian virus 40.

Viruses are remarkable self-assembled nanobiomaterial-based machines, exposed to a wide range of pH values. Extreme pH values can induce dramatic structural changes, critical for the function of the virus nanoparticles, including assembly and genome uncoating. Tuning cargo-capsid interactions is essential for designing virus-based delivery systems. Here we show how pH controls the structure and activity of wild-type simian virus 40 (wtSV40) and the interplay between its cargo and capsid. Using cryo-TEM and solution X-ray scattering, we found that wtSV40 was stable between pH 5.5 and 9, and only slightly swelled with increasing pH. At pH 3, the particles aggregated, while capsid protein pentamers continued to coat the virus cargo but lost their positional correlations. Infectivity was only partly lost after the particles were returned to pH 7. At pH 10 or higher, the particles were unstable, lost their infectivity, and disassembled. Using time-resolved experiments we discovered that disassembly began by swelling of the particles, poking a hole in the capsid through which the genetic cargo escaped, followed by a slight shrinking of the capsids and complete disassembly. These findings provide insight into the fundamental intermolecular forces, essential for SV40 function, and for designing virus-based nanobiomaterials, including delivery systems and antiviral drugs.

Studying viruses using solution X-ray scattering.

Viruses have been of interest to mankind since their discovery as small infectious agents in the nineteenth century. Because many viruses cause diseases to humans and agriculture, they were rigorously studied for biological and medical purposes. Viruses have remarkable properties such as the symmetry and self-assembly of their protein envelope, maturation into infectious virions, structural stability, and disassembly. Solution X-ray scattering can probe structures and reactions in solutions, down to subnanometer spatial resolution and millisecond temporal resolution. It probes the bulk solution and reveals the average shape and average mass of particles in solution and can be used to study kinetics and thermodynamics of viruses at different stages of their life cycle. Here we review recent work that demonstrates the capabilities of solution X-ray scattering to study in vitro the viral life cycle.

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.