Individual Protofilaments Research Articles

Protofilament-specific nanopatterns of tubulin post-translational modifications regulate the mechanics of ciliary beating

Controlling ciliary beating is essential for motility and signaling in eukaryotes. This process relies on the regulation of various axonemal proteins that assemble in stereotyped patterns onto individual microtubules of the ciliary structure. Additionally, each axonemal protein interacts exclusively with determined tubulin protofilaments of the neighboring microtubule to carry out its function. While it is known that tubulin post-translational modifications (PTMs) are important for proper ciliary motility, the mode and extent to which they contribute to these interactions remain poorly understood. Currently, the prevailing understanding is that PTMs can confer functional specialization at the level of individual microtubules. However, this paradigm falls short of explaining how the tubulin code can manage the complexity of the axonemal structure where functional interactions happen in defined patterns at the sub-microtubular scale. Here, we combine immuno-cryo-electron tomography (cryo-ET), expansion microscopy, and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nanopatterns at a sub-microtubular scale. These nanopatterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are indispensable for their regulation during ciliary beating. Our findings offer a new paradigm for understanding how different tubulin PTMs, such as glycylation, glutamylation, acetylation, tyrosination, and detyrosination, can coexist within the ciliary structure and specialize individual protofilaments for the regulation of diverse protein complexes. The identification of a ciliary tubulin nanocode by cryo-ET suggests the need for high-resolution studies to better understand the molecular role of PTMs in other cellular compartments beyond the cilium.

P-080 Unveiling the molecular determinants of sperm endpiece stabilization

Abstract Study question How are microtubules at the endpiece of the mammalian sperm tail stabilized? Summary answer SAX01 and SPACA9 are microtubule inner proteins that binds at the endpieces. In addition, singlet microtubules are capped by a complex of proteins. What is known already At the core of the sperm flagellum is the axoneme, a supramolecular assembly of nine doublet microtubules around a central pair of singlet microtubules. At the endpiece, axoneme structure is lost and only singlet microtubules remain. Using cryo-electron microscopy (cryo-EM) our group showed that the singlet microtubules contain species-specific microtubule inner proteins in a helical arrangement. Furthermore, we observed a protein density capping the microtubules plus-ends in pig, horse, and mouse sperm. Study design, size, duration To elucidate the function of the sperm endpiece plug and its associated proteins, we apply single-particle analysis cryo-EM. Participants/materials, setting, methods - Main results and the role of chance We revealed that the microtubule inner proteins in the singlets are SAX01 and SPACA9. Our high-resolution structure shows that SPACA9 forms a discontinuously intraluminal spiral. It consists of a bundle of alpha helices that binds across the intradimer α/β-tubulin interface as well as between adjacent protofilaments. Specifically, SPACA9 interacts with both α- and β-tubulin within a dimer while also interacting with the M-loop of α-tubulin from the neighbouring protofilament. Our structure further shows that SAX01 binds longitudinally along individual protofilaments with its Mn motif, a 5- to 6-residue helix flanked by a Tyr/Phe residue on one end and by a Ser/Thr on the other that binds the shoulder of the α -tubulin S9/S10 loop. We further observed that microtubule termini are capped with yet to be identified protein complexes. Taken together, we show that microtubule inner proteins longitudinally and laterally reinforce the microtubule lattice, while capped in their termini. Limitations, reasons for caution Limitations include species-specific variations as we previously observed between mouse sperm endpieces and porcine or horse. Cryo-EM structures represent averages across singlet microtubules, so individual variation within particles may be lost. Wider implications of the findings Investigating the mechanism behind stabilization of the endpiece singlet microtubules using cryo-EM provides better understanding of sperm tail stabilization that holds potential for progress in understanding male infertility and can be utilized for a development of male contraception. Trial registration number not applicable

Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging.

Microtubule structure is commonly investigated using single-particle analysis (SPA) or subtomogram averaging (STA), whose main objectives are to gather high-resolution information on the αβ-tubulin heterodimer and on its interactions with neighboring molecules within the microtubule lattice. The maps derived from SPA approaches usually delineate a continuous organization of the αβ-tubulin heterodimer that alternate regularly head-to-tail along protofilaments, and that share homotypic lateral interactions between monomers (α-α, β-β), except at one unique region called the seam, made of heterotypic ones (α-β, β-α). However, this textbook description of the microtubule lattice has been challenged over the years by several studies that revealed the presence of multi-seams in microtubules assembled in vitro from purified tubulin. To analyze in deeper detail their intrinsic structural heterogeneity, we have developed a segmented subtomogram averaging (SSTA) strategy on microtubules decorated with kinesin motor-domains that bind every αβ-tubulin heterodimer. Individual protofilaments and microtubule centers are modeled, and sub-volumes are extracted at every kinesin motor domain position to obtain full subtomogram averages of the microtubules. The model is divided into shorter segments, and subtomogram averages of each segment are calculated using the main parameters of the full-length microtubule settings as a template. This approach reveals changes in the number and location of seams within individual microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts. Key features This protocol builds upon the method developed by J.M. Heumann to perform subtomogram averages of microtubules and extends it to divide them into shorter segments. Microtubules are decorated with kinesin motor-domains to determine the underlying organization of its constituent αβ-tubulin heterodimers. The SSTA approach allows analysis of the structural heterogeneity of individual microtubules and reveals multi-seams and changes in their number and location within their shaft. Graphical overview.

Nanoscale Structural Analysis of a Lipid-Driven Aggregation of Insulin.

Abrupt aggregation of misfolded proteins is a hallmark of a large number of severe pathologies, including diabetes types 1 and 2, Alzheimer, and Parkinson diseases. A growing body of evidence suggests that lipids can uniquely change rates of amyloid-associated proteins as well as modify the structure of formed oligomers and fibrils. In this study, we utilize atomic force microscopy infrared (AFM-IR) spectroscopy, also known as nano-IR spectroscopy, to examine the structure of individual insulin oligomers, protofilaments, and fibrils grown in the presence of phospholipids. Our findings show that AFM-IR spectra of insulin oligomers have strong signals of C-H and PO2- vibrations, which points on the presence of lipids in the oligomer structure. Furthermore, substantial shifts in lipid vibrations in AFM-IR spectra of the oligomers relative to the corresponding bands of pure lipids have been observed. This points on strong interactions between a lipid and a protein that are developed at the stage of the oligomer formation.

Bending-torsional elasticity and energetics of the plus-end microtubule tip

Microtubules (MTs), mesoscopic cellular filaments, grow primarily by the addition of GTP-bound tubulin dimers at their dynamic flaring plus-end tips. MTs can perform mechanical work by cycling between phases of growth and shortening, driven by both GTP hydrolysis in tubulin subunits and dynamics of individual protofilaments (PFs) at the plus-end tips. The mechanochemical basis of energy conversion and transduction by MTs has remained elusive and controversial, despite extensive work. Moreover, recent experimental findings have created the paradox that the plus-end MT tips look very similar during both growing and shrinking phases, thereby challenging the traditional textbook picture.

Measuring Microtubule Supertwist and Defects by Three-Dimensional-Force-Clamp Tracking of Single Kinesin-1 Motors.

Three-dimensional (3D) nanometer tracking of single biomolecules provides important information about their biological function. However, existing microscopy approaches often have only limited spatial or temporal precision and do not allow the application of defined loads. Here, we developed and applied a high-precision 3D-optical-tweezers force clamp to track in vitro the 3D motion of single kinesin-1 motor proteins along microtubules. To provide the motors with unimpeded access to the whole microtubule lattice, we mounted the microtubules on topographic surface features generated by UV-nanoimprint lithography. Because kinesin-1 motors processively move along individual protofilaments, we could determine the number of protofilaments the microtubules were composed of by measuring the helical pitches of motor movement on supertwisted microtubules. Moreover, we were able to identify defects in microtubules, most likely arising from local changes in the protofilament number. While it is hypothesized that microtubule supertwist and defects can severely influence the function of motors and other microtubule-associated proteins, the presented method allows for the first time to fully map the microtubule lattice in situ. This mapping allows the correlation of motor-filament interactions with the microtubule fine-structure. With the additional ability to apply loads, we expect our 3D-optical-tweezers force clamp to become a valuable tool for obtaining a wide range of information from other biological systems, inaccessible by two-dimensional and/or ensemble measurements.

Accounting for Microtubule Distortions in Cryo-EM Structures using Patch Refinements

Microtubules are dynamic, filamentous structures that are essential for many different cellular processes. These processes often rely on a number of microtubule associated proteins (MAPs) that either regulate or carry out these functions. Characterizing interactions between MAPs and microtubules is crucial to understanding of how these processes are carried out. Recent advances in cryo-electron microscopy have made observing interactions between microtubules and several different MAPs at near atomic resolution possible. However, the resolution of these structures is variable, depending on the MAP. Recent studies have shown that the microtubule lattice is flexible and different stabilizing drugs and MAPs affects this flexibility. These results suggest that distortions in the microtubule lattice are limiting overall resolution. Rather than aligning whole segments of microtubules (like in conventional microtubule reconstructions), we have developed a technique that aligns patches of tubulin. By subtracting the surrounding microtubule density and leaving a patch of tubulin dimers, each patch can be aligned independently from the bulk of the microtubule. We have tested this ‘patch refinement’ technique on a synthetically, distorted dataset. Using traditional reconstruction techniques yielded a low resolution structure, but upon application of the patch refinement technique, the resulting structure was nearly indistinguishable from an equivalent undistorted, synthetic dataset. The patch refinement method has also been applied to an experimental dataset that was resolved to >5Å using traditional methods. Following patch refinement, the resolution increased to <3.6Å. Using the alignment parameters following patch refinement, we were able to trace the path of individual protofilaments and observe distortions in the lattice. Further use and development of this technique will allow us to analyze microtubule distortions and work towards obtaining an atomic structure of a microtubule.

Self-Organization of FtsZ Polymers in Solution Reveals Spacer Role of the Disordered C-Terminal Tail

FtsZ is a self-assembling GTPase that forms, below the inner membrane, the mid-cell Z-ring guiding bacterial division. FtsZ monomers polymerize head to tail forming tubulin-like dynamic protofilaments, whose organization in the Z-ring is an unresolved problem. Rather than forming a well-defined structure, FtsZ protofilaments laterally associate in vitro into polymorphic condensates typically imaged on surfaces. We describe here nanoscale self-organizing properties of FtsZ assemblies in solution that underlie Z-ring assembly, employing time-resolved x-ray scattering and cryo-electron microscopy. We find that FtsZ forms bundles made of loosely bound filaments of variable length and curvature. Individual FtsZ protofilaments further bend upon nucleotide hydrolysis, highlighted by the observation of some large circular structures with 2.5–5° curvature angles between subunits, followed by disassembly end-products consisting of highly curved oligomers and 16-subunit −220 Å diameter mini-rings, here observed by cryo-electron microscopy. Neighbor FtsZ filaments in bundles are laterally spaced 70 Å, leaving a gap in between. In contrast, close contact between filament core structures (∼50 Å spacing) is observed in straight polymers of FtsZ constructs lacking the C-terminal tail, which is known to provide a flexible tether essential for FtsZ functions in cell division. Changing the length of the intrinsically disordered C-tail linker modifies the interfilament spacing. We propose that the linker prevents dynamic FtsZ protofilaments in bundles from sticking to one another, holding them apart at a distance similar to the lateral spacing observed by electron cryotomography in several bacteria and liposomes. According to this model, weak interactions between curved polar FtsZ protofilaments through their the C-tails may facilitate the coherent treadmilling dynamics of membrane-associated FtsZ bundles in reconstituted systems, as well as the recently discovered movement of FtsZ clusters around bacterial Z-rings that is powered by GTP hydrolysis and guides correct septal cell wall synthesis and cell division.

Termination of Protofilament Elongation by Eribulin Induces Lattice Defects that Promote Microtubule Catastrophes

Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation ofindividual protofilaments can potentially lead toan altered microtubule-end structure that promotessudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation.

Three-Dimensional Motility of the Highly Processive Kinesin-8 Along the Microtubule Lattice

During mitosis, kinesin-8 motors play a key role in regulating the spindle length based to their depolymerization activity at microtubule plus-ends. In order to reach the plus-ends of microtubules, kinesin-8 motors show superprocessive motility. However, the mechanism confering such high motor processivity even on crowded microtubules in the cytoplasm is not well understood. To gain insight into the stepping mechanism of kinesin-8, we explored the three-dimensional motility of yeast kinesin-8, Kip3, along the microtubule lattice in vitro. First, we performed microtubule gliding motility assays on surface coated with Kip3 motors and measured the rotations of gliding microtubules around their longitudinal axis using rhodamine speckled microtubules in combination with fluorescence-interference contrast microscopy [1]. We observed rotations with periodicities significantly smaller than the microtubule supertwist, indicating that the motors do not follow individual protofilaments but rather switch protofilaments stochastically in one direction [2]. Finally, we confirmed this hypothesis by 3D single-molecule stepping assays along freely suspended microtubules by 2D tracking of QDot-conjugated Kip3 motors in combination with parallax [3], a dual-focus imaging technique that provides nanometer information in z-direction.1. Mitra A, Ruhnow F, Nitzsche B, Diez S. Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces. PLoS One. 2015.2. Bormuth V, Nitzsche B, Ruhnow F, Mitra A, Storch M, Rammner B, Howard J, Diez S. The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left. Biophys J. 2012.3. Sun Y, McKenna JD, Murray JM, Ostap EM, Goldman YE. Parallax: high accuracy three-dimensional single molecule tracking using split images. Nano Lett. 2009.

Microtubule‐Binding R3 Fragment from Tau Self‐Assembles into Giant Multistranded Amyloid Ribbons

AbstractTau protein and its fragments self‐assemble into amyloid fibrils in the presence of polyanions, such as heparin. By combining microscopy, scattering, and spectroscopy techniques, we studied the aggregation of the 26‐mer Tau‐derived peptide alone, Tau306–327, the third repeat fragment (R3) of the microtubule‐binding domain. We show that: i) the sole Tau306–327 can self‐assemble into amyloid fibrils without the need of aggregation‐promoting polyanions; ii) the resulting structures consist of surprisingly large, well‐ordered 2D laminated flat ribbons, with a log‐normal distribution of the lateral width, reaching the unprecedented lateral size of 350 nm and/or 45 individual protofilaments, that is, the largest amyloid laminated structures ever observed for Tau or any other amyloidogenic sequence. Our results provide insight into the molecular determinants of Tau aggregation and open new perspectives in the understanding of the assembly of amyloid fibrils and β‐sheet‐based biomaterials.

Microtubule-Binding R3 Fragment from Tau Self-Assembles into Giant Multistranded Amyloid Ribbons.

Tau protein and its fragments self-assemble into amyloid fibrils in the presence of polyanions, such as heparin. By combining microscopy, scattering, and spectroscopy techniques, we studied the aggregation of the 26-mer Tau-derived peptide alone, Tau(306-327), the third repeat fragment (R3) of the microtubule-binding domain. We show that: i) the sole Tau(306-327) can self-assemble into amyloid fibrils without the need of aggregation-promoting polyanions; ii) the resulting structures consist of surprisingly large, well-ordered 2D laminated flat ribbons, with a log-normal distribution of the lateral width, reaching the unprecedented lateral size of 350 nm and/or 45 individual protofilaments, that is, the largest amyloid laminated structures ever observed for Tau or any other amyloidogenic sequence. Our results provide insight into the molecular determinants of Tau aggregation and open new perspectives in the understanding of the assembly of amyloid fibrils and β-sheet-based biomaterials.

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer’s. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor’s specific intracellular tasks and environments.

Collective Force Generation by Microtubules and Kinetochores

A microtubule (MT) is a stiff nanotube that, normally, consists of 13 protofilaments. During polymerization, it generates pushing force against a light obstacle and operationally resembles a nano-piston. Similarly a depolymerizing MT operates as a nano-hook generating a pulling force. During mitosis in eukaryotic cells each of the two sister chromatids, that tightly embrace each other, is bound to a separate proteinous structure called kinetochore (kt). One end of the kinetochore fiber that, in general, consists of several MTs, attaches with a single kt and exerts a poleward pull on it while the MTs depolymerize. Budding yeast is special in the sense that each kt attaches to only a single MT. Two oppositely oriented MTs pull the two kts attached to two sister chromatids thereby pulling the two sisters away from each other in the anaphase. In one of our earlier publications, we theoretically studied a simple in-vitro system consisting of a single MT coupled to an isolated kt that is subjected to an external tension. Although the theory could account for the observed catch-bond-like behavior of the kt-MT attachment, the kinetics of the individual protofilaments were not treated explicitly. In this presentation we show the collective effects of polymerization and depolymerization of the individual protofilaments of the MT. Extending the model even further we explore the collective effects of multiple MTs in a kinetochore fiber attached to a single kt. Our theory provides a deep insight into the effects of (a) structure, (b) energetics, and (c) stochastic kinetics of the kt-MT coupling on the collective force-velocity relation of these attachments.

Brownian Dynamics of Subunit Addition-Loss Kinetics and Thermodynamics in Linear Polymer Self-Assembly

The structure and free energy of multistranded linear polymer ends evolves as individual subunits are added and lost. Thus, the energetic state of the polymer end is not constant, as assembly theory has assumed. Here we utilize a Brownian dynamics approach to simulate the addition and loss of individual subunits at the polymer tip. Using the microtubule as a primary example, we examined how the structure of the polymer tip dictates the rate at which units are added to and lost from individual protofilaments. We find that freely diffusing subunits arrive less frequently to lagging protofilaments but bind more efficiently, such that there is no kinetic difference between leading and lagging protofilaments within a tapered tip. However, local structure at the nanoscale has up to an order-of-magnitude effect on the rate of addition. Thus, the kinetic on-rate constant, integrated across the microtubule tip (kon,MT), is an ensemble average of the varying individual protofilament on-rate constants (kon,PF). Our findings have implications for both catastrophe and rescue of the dynamic microtubule end, and provide a subnanoscale framework for understanding the mechanism of action of microtubule-associated proteins and microtubule-directed drugs. Although we utilize the specific example of the microtubule here, the findings are applicable to multistranded polymers generally.

In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy.

In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule-based super-resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments.

Dynein Tethers and Stabilizes Dynamic Microtubule Plus Ends

Microtubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The "search and capture" model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains. Whereas cytoplasmic dynein is primarily known as a minus-end-directed microtubule motor for organelle transport, cortically localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP dependent, indicating that dynein motor activity was required. Computational modeling suggests that dynein delays catastrophe by exerting tension on individual protofilaments, leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus ends.

Snapshots of fibrillation and aggregation kinetics in multistranded amyloid β-lactoglobulin fibrils

We have investigated the structural time-evolution of multistranded β-lactoglobulin protein fibrils at pH 2 and 90 °C by combining small angle neutron scattering (SANS), dynamic (DLS) and depolarized light scattering (DDLS) as well as atomic force microscopy (AFM). Light scattering techniques, combined with SANS clearly demonstrate the different stages of conversion of β-lactoglobulin monomers (2 wt %) into semiflexible protein fibrils upon heating at 90 °C. In addition, atomic force microscopy allows the resolution of some details of the fibrils at the molecular length scale which bulk scattering techniques cannot capture. Thus, we were able to resolve and identify different individual stages of the fibrillation process, including the formation of protofilaments, their alignment and aggregation into mature multistranded fibrils, and the development of a periodic pitch along their contour length. The picture emerging from combination of the scattering and single molecule techniques is consistent with three critical steps: (i) individual protofilaments align upon approaching due to liquid crystalline interactions; (ii) short range attractions among filaments—presumably of Lennard–Jones or hydrophobicity type—lead to irreversible aggregation of nearly perfectly aligned protofilaments into multistranded ribbon-like fibrils; (iii) intramolecular electrostatic repulsion of fibrils leads to twisting of the ribbon along the axis leading to the development of a periodic pitch along the fibrils contour length. The individual stages of the fibrillation and aggregation process are discussed in detail, in terms of the colloidal physics involved.

X-Ray Fiber Diffraction Reveals Major Structural Differences Between Brain-Derived Prions and Recombinant Prion Protein Amyloid

X-ray fiber diffraction was used to study the structure of brain-derived prions and recombinant protein amyloid. Partially oriented, dried fibers were prepared from brain-derived PrP 27-30 and recombinant PrP amyloid. Fiber diffraction patterns were analyzed and used to interrogate models for the structure of the infectious prion.Fiber diffraction patterns of recombinant PrP amyloid displayed characteristic, meridional reflections at ∼4.8 A and equatorial reflections at ∼10 A. These patterns were similar to those of other amyloids and are consistent with a basic cross-β architecture. In contrast, diffraction patterns from brain-derived PrP 27-30 displayed meridional reflections at ∼9.6, ∼6.4, and ∼4.8 A, which correspond to the 2nd, 3rd, and 4th order of a ∼19.2-A repeating unit, suggesting that PrP 27-30 contains four β-strands in a cross-β architecture. Furthermore, PrP 27-30 lacked the typical, equatorial reflection at ∼10 A, but instead produced equatorial reflections characterizing the diameter of the amyloid fiber and of individual protofilaments. Therefore, PrP 27-30 seems to have a structure consistent with a β-helix or β-solenoid, not unlike the model that was proposed earlier (Govaerts et al., 2004). This interpretation was also supported by extensive modeling, simulation of diffraction, electron microscopy, and FTIR.In a previous study, recombinant PrP amyloid induced a transmissible disease in transgenic mice overexpressing PrP, and was thus termed a prion (Legname et al., 2004). Serially transmitted, synthetic prions were purified from mouse brains and analyzed by fiber diffraction. These brain-derived, synthetic prions showed the same structural characteristics as natural isolates and not those of its recombinant protein precursor. The relationship between structural differences and infectivity can be explained by several hypotheses. It remains to be determined which one, if any, is correct.

A theory of microtubule catastrophes and their regulation

Dynamic instability, in which abrupt transitions occur between growing and shrinking states, is an intrinsic property of microtubules that is regulated by both mechanics and specialized proteins. We discuss a model of dynamic instability based on the popular idea that growth is maintained by a cap at the tip of the fiber. The loss of this cap is thought to trigger the transition from growth to shrinkage, called a catastrophe. The model includes longitudinal interactions between the terminal tubulins of each protofilament and "gating rescues" between neighboring protofilaments. These interactions allow individual protofilaments to transiently shorten during a phase of overall microtubule growth. The model reproduces the reported dependency of the catastrophe rate on tubulin concentration, the time between tubulin dilution and catastrophe, and the induction of microtubule catastrophes by walking depolymerases. The model also reproduces the comet tail distribution that is characteristic of proteins that bind to the tips of growing microtubules.