Nucleation-dependent tau filament formation: the importance of dimerization and an estimation of elementary rate constants

Filamentous inclusions composed of the microtubule-associated protein tau are found in Alzheimer disease and other tauopathic neurodegenerative diseases, but the mechanisms underlying their formation from full-length protein monomer under physiological conditions are unclear. To address this issue, the fibrillization of recombinant full-length four-repeat human tau was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods and a small-molecule inducer of aggregation, thiazine red. Data were then fit to a simple homogeneous nucleation model with rate constant constraints established from filament dissociation rate, critical concentration, and mass-per-unit length measurements. The model was then tested by comparing the predicted time-dependent evolution of length distributions to experimental data. Results indicated that once assembly-competent conformations were attained, the rate-limiting step in the fibrillization pathway was tau dimer formation. Filament elongation then proceeded by addition of tau monomers to nascent filament ends. Filaments isolated at reaction plateau contained approximately 2 tau protomers/beta-strand spacing on the basis of mass-per-unit length measurements. The model suggests four key steps in the aggregation pathway that must be surmounted for tau filaments to form in disease.     

Cyanine dye N744 inhibits tau fibrillization by blocking filament extension: implications for the treatment of tauopathic neurodegenerative diseases

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Small molecules capable of both inhibiting aggregation and promoting filament disaggregation have been discovered, but knowledge of their mechanism of action and potential for testing in biological models is fragmentary. To clarify these issues, the interaction between a small-molecule inhibitor of tau fibrillization, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), and full-length four-repeat tau protein was characterized in vitro using transmission electron microscopy and fluorescence spectroscopy. Analysis of reaction time courses performed in the presence of anionic fibrillization inducers revealed that increasing concentrations of N744 decreased the total filament length without modulating lag time, indicating that filament extension but not nucleation was affected by inhibitor under the conditions that were investigated. Critical concentration measurements confirmed that N744 shifted equilibria at filament ends away from the fibrillized state, resulting in endwise filament disaggregation when it was added to synthetic filaments. Both increasing bulk tau concentrations and filament stabilizing modifications such as pseudophosphorylation and glycation antagonized N744 activity. The results illustrate the importance of mechanism for the design and interpretation of pharmacological studies in biological models of tau aggregation.     

Spatial and temporal variability in filament length of a toxic cyanobacterium (Anabaena affinis)

1. This study examines the distribution of Anabaena affinis filament lengths under natural conditions as a function of depth and season, and in the laboratory as a function of growth phase. Because Anabaena affinis is only toxic when consumed, both its filament length and position in the water column are important determinants of its potential impact on zooplankton populations. 2. Star Lake (Norwich, Vermont, U.S.A.), a natural, eutrophic pond, remained thermally stratified throughout the Anabaena bloom. Filament number and length differed significantly with both sampling date and water depth. Most filaments occurred at 0.5 m, particularly at the height of the bloom. Throughout the entire water column average filament length decreased from approximately 0.53 mm in May to 0.14 mm in July. The shortest filaments occurred at the 2.5 m depth. Filament length distributions (combined for all depths) for 29 May, 12 June and 3 July, corresponding to the beginning, middle and end of the bloom, respectively, differed significantly among the three dates. These patterns most likely reflect variable growth conditions, both during the season and in the water column. 3. In the laboratory, Anabaena filament length was affected by medium composition and growth phase. Filaments were significantly longer when grown in MBL than in ASM medium. Also, the average length of Anabaena filaments grown in MBL changed significantly as cultures aged; by day 13 filament length (2.01 ± 0.38 mm, mean ± SD) was twice that on day 0 (0.97 ± 0.71 mm). As cell concentration continued to increase, mean filament length gradually decreased.     

Kinetic mechanism of end-to-end annealing of actin filaments

We investigated the effect of actin filament length and capping protein on the rate of end-to-end annealing of actin filaments. Long filaments were fragmented by shearing and allowed to recover. Stabilizing filaments with phalloidin in most experiments eliminated any contribution of subunit dissociation and association to the redistribution of lengths but did not affect the results. Two different assays, fluorescence microscopy to measure filament lengths and polymerization to measure concentration of barbed filament ends, gave the same time-course of annealing. The rate of annealing declines with time as the average filament length increases. Longer filaments also anneal slower than short filaments. The second-order annealing rate constant is inversely proportional to mean polymer length with a value of 1.1 mM(-1) s(-1)/length in subunits. Capping protein slows but does not prevent annealing. Annealing is a highly favorable reaction with a strong influence on the length of polymers produced by spontaneous polymerization and should be considered in thinking about polymer dynamics in cells.     

Site-specific nitration differentially influences tau assembly in vitro

Previously, we reported that the microtubule-associated tau protein, the major constituent of neurofibrillary tangles (NFTs) in Alzheimer's brain, undergoes site-selective nitration by peroxynitrite (ONOO-) and that this event inhibits tau polymerization in vitro [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we extend our analysis of tau nitration to include mutant tau proteins singly nitrated at each residue targeted by ONOO- in vitro (Tyr18, Tyr29, Tyr197, and Tyr394). Using our polymerization paradigm, we demonstrate that site-specific Tyr nitration differentially alters the rate and/or extent of tau assembly and generates robust changes in filament morphology. As determined by quantitative electron microscopy, select nitration of residues Tyr29 and Tyr197 increases the average length of synthetic tau filaments but does not alter the steady-state polymer mass. In contrast, site-specific nitration of residues Tyr18 and Tyr394 decreases the average length and/or number of synthetic filaments, resulting in a significant reduction in filamentous mass and an increase in tau critical concentration. Intriguingly, affinity measurements demonstrate that nitrative modifications do not preclude formation of the Alz-50 epitope, a pathological tau conformation detectable in authentic paired helical filaments (PHFtau). In fact, the Alz-50 antibody binds filaments assembled from nitrated mutant tau with higher avidity than wild-type filaments, even in instances where the overall filamentous mass is reduced. Taken together, our results suggest that site-specific nitration modulates the nucleation and/or elongation capacity of assembly-competent tau and that assumption of the Alz-50 conformation may be necessary, but not sufficient, to induce filament formation.     

FILAMENT LENGTHS IN STRIATED MUSCLE

Filament lengths in resting and excited frog muscles have been measured in the electron microscope, and investigations made of the changes in length that are found under different conditions, to distinguish between those changes which arise during preparation and the actual differences in the living muscles. It is concluded that all the measured differences in filament length are caused by the preparative procedures in ways that can be simply accounted for, and that the filament lengths are the same in both resting and excited muscles at all sarcomere lengths greater than 2.1 µ, viz., A filaments, 1.6 µ; I filaments, 2.05 µ. The fine periodicity visible along the I filaments also has been measured in frog, toad, and rabbit muscles and found to be 406 A.     

Ligand-dependent tau filament formation: implications for Alzheimer's disease progression.

The mechanism through which arachidonic acid induces the polymerization of tau protein into filaments under reducing conditions was characterized through a combination of fluorescence spectroscopy and electron microscopy. Results show that polymerization follows a ligand-mediated mechanism, where binding of arachidonic acid is an obligate step preceding tau-tau interaction. Homopolymerization begins with rapid (on the order of seconds) nucleation, followed by a slower elongation phase (on the order of hours). Although essentially all synthetic filaments have straight morphology at early time points, they interact with thioflavin-S and monoclonal antibody Alz50 much like authentic paired helical filaments, suggesting that the conformation of tau protein is similar in the two filament forms. Over a period of days, synthetic straight filaments gradually adopt paired helical morphology. These results define a novel pathway of tau filament formation under reducing conditions, where oxidation may contribute to final paired helical morphology, but is not a necessary prerequisite for efficient nucleation or elongation of tau filaments.     

Ligand-dependent inhibition and reversal of tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule associated protein tau. Because animal model studies suggest that a toxic gain of function accompanies tau aggregation in neurons, selective pharmacological inhibitors of the process may have utility in slowing neurodegeneration. Here, the properties of a candidate small molecule inhibitor of tau fibrillization, 3-(2-hydroxyethyl)-2-[2-[[3-(2-hydroxyethyl)-5-methoxy-2-benzothiazolylidene]methyl]-1-butenyl]-5-methoxybenzothiazolium (N744), were characterized in vitro using transmission electron microscopy. N744 inhibited arachidonic acid-induced aggregation of full-length, four-repeat tau protein at substoichiometric concentrations relative to total tau and with an IC(50) of approximately 300 nM. Inhibition was accompanied by a dose-dependent decrease in the number concentration of filaments, suggesting that N744 interfered with tau filament nucleation. Stoichiometric concentrations of N744 also promoted tau disaggregation when added to mature synthetic filaments. Disaggregation followed first-order kinetics and was accompanied by a steady decrease in filament number, suggesting that N744 promoted endwise loss of tau molecules with limited filament breakage. N744 at substoichiometric concentrations did not inhibit Abeta and alpha-synuclein aggregation, indicating it was tau selective under these conditions. Because of its activity in vitro, N744 may offer a pharmacological approach to the role of tau fibrillization in neurodegeneration.     

EMASCULATION EFFECTS ON THE STAMEN FILAMENT OF NIGELLA HTSPANICA AND THEIR PARTIAL REVERSAL BY GIBBERELLIC ACID

Anther removal from stamens whose filaments are 1–3 mm long restricts filament elongation to approximately 60% of the normal length. Removal of one-third to one-half of the anthers affects only the antherless filaments and does not appear to inhibit the growth of the other organs of the flower. Filament growth inhibition induced by anther removal involves both an inhibition of epidermal cell elongation along the length of the filament and also an inhibition of cell division. There is no evidence that the inhibition of filament growth is a response to damage caused by anther removal. Rather, it is suggested that anther removal severs a normal hormonal relationship existing between the anther and the developing filament. The application of gibberellic acid (GA₃) in lanolin paste stimulated the elongation of the antherless filaments to achieve an average of 87% of the filament length of adjacent intact stamens. The closer a filament is to having attained its final number of cells before anther removal, the closer does its length come to reaching the final length of filaments in intact stamens. The elongation of these antherless filaments with the application of GA₃ was accompanied by elongation of the epidermal cells of the filament to normal, or in some cases greater than normal, lengths. There is no evidence that GA₃ application affected the inhibition of epidermal cell devision induced by anther removal. The results of this study support the suggestion of Plack that emasculation-induced inhibition in the growth of floral organs and its reversal by GA₃ is a general phenomenon.     

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.     

Spatiotemporal Dynamics of Actomyosin Networks

Rhodamine–phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ∼40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ∼13 k_BT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an “order parameter,” peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes.     

Spatiotemporal Dynamics of Actomyosin Networks

Rhodamine–phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ∼40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ∼13 k_BT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an “order parameter,” peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes.     

Myosin Motors Drive Long Range Alignment of Actin Filaments

The bulk alignment of actin filament sliding movement, powered by randomly oriented myosin molecules, has been observed and studied using an in vitro motility assay. The well established, actin filament gliding assay is a minimal experimental system for studying actomyosin motility. Here, we show that when the assay is performed at densities of actin filaments approaching those found in living cells, filament gliding takes up a preferred orientation. The oriented patterns of movement that we have observed extend over a length scale of 10–100 μm, similar to the size of a mammalian cell. We studied the process of filament alignment and found that it depends critically upon filament length and density. We developed a simple quantitative measure of filament sliding orientation and this enabled us to follow the time course of alignment and the formation and disappearance of oriented domains. Domains of oriented filaments formed spontaneously and were separated by distinct boundaries. The pattern of the domain structures changed on the time scale of several seconds and the collision of neighboring domains led to emergence of new patterns. Our results indicate that actin filament crowding may play an important role in structuring the leading edge of migrating cells. Filament alignment due to near-neighbor mechanical interactions can propagate over a length scale of several microns; much greater than the size of individual filaments and analogous to a log drive. Self-alignment of actin filaments may make an important contribution to cell polarity and provide a mechanism by which cell migration direction responds to chemical cues.     

Characterization of villin from the intestinal brush border of the rat, and comparative analysis with avian villin

The biochemical properties of villin purified from the brush borders of chicken and rat small intestines were compared, with emphasis on their physical properties and their Ca++-dependent interaction with actin. Like chicken villin, rat villin exists as two isoforms present in equimolar concentrations; the rat isoforms are slightly more acidic than those of chicken villin (6.08 and 6.11 versus 6.26 and 6.34). Rabbit antisera raised against either villin crossreacted with the other one. Like the avian protein, rat villin bundled F-actin at calcium concentrations below 0.1 microM. Above approximately 1 microM calcium, it accelerated the rate of actin assembly and restricted filament lengths of F-actin formed either during coassembly with villin or by addition of villin to preformed filaments. The threshold calcium concentration required for effective severing of preformed filaments was approximately tenfold higher than that required for restricting lengths during coassembly. The extent of filament shortening was proportional to the amount of villin present. At a fixed villin concentration, filament length decreased with increasing [Ca++] over a broad range from 10(-7)-10(-4) M. In general, the mean filament lengths and the dispersion about the mean value were lower in samples where filaments were coassembled with villin than when villin was added to preformed filaments.     

Structure and mobility of actin filaments as measured by quasielastic light scattering, viscometry, and electron microscopy

Actin filaments of different lengths were prepared by polymerizing actin in the presence of various concentrations of gelsolin, a protein which accelerates actin polymerization by stabilizing nuclei from which filaments grow and which binds to their fast growing ends. The lengths of the actin filaments following polymerization were measured by electron microscopy and showed that the number-average filament length agreed with the predicted length if each gelsolin molecule acted as a seed for the growth of an actin filament. The distribution of lengths was independent of the actin:gelsolin ratio and was similar to that of actin filaments polymerized in the absence of gelsolin (Lw/Ln = 1.8). The mobility of these filaments in solution was studied by quasielastic light scattering and by viscometry. The translational diffusion constant determined by quasielastic light scattering was in agreement with the infinite dilution values calculated from the dimensions and the distribution of lengths determined by electron microscopy for relatively short filament lengths. Under conditions where overlap of the rotational domains of the filaments would be expected to occur, the measured diffusion rates deviated from their predicted dilute solution values and the solution viscosity increased abruptly. The dependence of the diffusion constant and the solution viscosity on the length of the actin filaments can be explained in terms of a theory that describes the restraints on diffusion of independent rigid rods in semi-dilute solution. The results suggest that the rheology of actin filaments can be accounted for by steric restraints. The length of cytoplasmic actin filaments in some cell types is such that these steric constraints are significant and could produce large changes in physical properties with small changes in filament length.     

Microtubule-dependent transport and dynamics of vimentin intermediate filaments

We studied two aspects of vimentin intermediate filament dynamics—transport of filaments and subunit exchange. We observed transport of long filaments in the periphery of cells using live-cell structured illumination microscopy. We studied filament transport elsewhere in cells using a photoconvertible-vimentin probe and total internal reflection microscopy. We found that filaments were rapidly transported along linear tracks in both anterograde and retrograde directions. Filament transport was microtubule dependent but independent of microtubule polymerization and/or an interaction with the plus end–binding protein APC. We also studied subunit exchange in filaments by long-term imaging after photoconversion. We found that converted vimentin remained in small clusters along the length of filaments rather than redistributing uniformly throughout the network, even in cells that divided after photoconversion. These data show that vimentin filaments do not depolymerize into individual subunits; they recompose by severing and reannealing. Together these results show that vimentin filaments are very dynamic and that their transport is required for network maintenance.     

Hydrostatic pressure studies of native and synthetic thick filaments: II. Native thick filaments from rabbit skeletal muscle

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14,000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at -20 degrees C for 6 months. Instead, filament dissociation proceeds linearly as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.     

Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends

Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concentration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more extensive severing by cofilin.     

The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.     

Disruption of the keratin filament network during epithelial cell division

The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle.