Real-Time 3D Imaging and Inhibition Analysis of Various Amyloid Aggregations Using Quantum Dots

Amyloidosis refers to aggregates of protein that accumulate and are deposited as amyloid fibrils into plaques. When these are detected in organs, they are the main hallmark of Alzheimer’s disease, Parkinson’s disease, and other related diseases. Recent medical advances have shown that many precursors and proteins can induce amyloidosis even though the mechanism of amyloid aggregation and the relationship of these proteins to amyloidosis remains mostly unclear. In this study, we report the real-time 3D-imaging and inhibition analysis of amyloid B (AB ), tau, and a-synuclein aggregation utilizing the a nity between quantum dots (QD) and amyloid aggregates. We successfully visualized these amyloid aggregations in real-time using fluorescence microscopy and confocal microscopy simply by adding commercially available QD. The observation by transmission electron microscopy (TEM) showed that QD particles bound to all amyloid fibrils. The 3D-imaging with QD revealed di erences between amyloid aggregates composed of di erent amyloid peptides that could not be detected by TEM.We were also able to quantify the inhibition activities of these proteins by rosmarinic acid, which has high activity for AB aggregation, from fluorescence micrographs as half-maximal e ective concentrations. These imaging techniques with QD serve as quick, easy, and powerful tools to understand amyloidosis and to discover drugs for therapies

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human alpha-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants

Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells

K336I mutant actin alters the structure of neighbouring protomers in filaments and reduces affinity for actin-binding proteins

Abstract Mutation of the Lys-336 residue of actin to Ile (K336I) or Asp (K336E) causes congenital myopathy. To understand the effect of this mutation on the function of actin filaments and gain insight into the mechanism of disease onset, we prepared and biochemically characterised K336I mutant actin from Dictyostelium discoideum. Subtilisin cleavage assays revealed that the structure of the DNase-I binding loop (D-loop) of monomeric K336I actin, which would face the adjacent actin-protomer in filaments, differed from that of wild type (WT) actin. Although K336I actin underwent normal salt-dependent reversible polymerisation and formed apparently normal filaments, interactions of K336I filaments with alpha-actinin, myosin II, and cofilin were disrupted. Furthermore, co-filaments of K336I and WT actins also exhibited abnormal interactions with cofilin, implying that K336I actin altered the structure of the neighbouring WT actin protomers such that interaction between cofilin and the WT actin protomers was prevented. We speculate that disruption of the interactions between co-filaments and actin-binding proteins is the primary reason why the K336I mutation induces muscle disease in a dominant fashion

The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

<div><p>The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.</p></div

FRET distributions.

<p>(A) Upper and lower panels show histograms of the FRET efficiencies of control FRET actin (red) and G146V FRET actin (blue), respectively. Assays were performed in the absence (left) and in the presence of sk MII HMM (middle) or MV HMM (right). The dashed lines and thick lines show each component of the fitted Gaussian distributions and their sum, respectively. The number of data points subjected to FRET analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126262#pone.0126262.s003" target="_blank">S1 Table</a>. (B) Changes in mean values calculated from overall FRET efficiencies induced by interaction with HMM. Statistical significance was assessed through the Mann—Whitney U-test. Asterisks indicate statistical significance (p < 0.00001). Error bars show SEM. All assays were performed in the presence of 1 mM ATP and the ATP regeneration system.</p

Parameters of depolymerization.

<p>Parameters were obtained by fitting data in Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126262#pone.0126262.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126262#pone.0126262.g001" target="_blank">1B</a> with double exponentials. The parameters ƒ_<i>s</i> and τ_<i>s</i> respectively give the fraction and depolymerization rate of the slow-depolymerization population, and τ_<i>f</i> gives the depolymerization rate of the fast-depolymerization population. Mean and standard deviation of three independent measurements are shown.</p><p>Parameters of depolymerization.</p

Polymerization and depolymerization of G146V and WT filaments.

<p>A 2 μM solution of monomeric actin (20% labeled with pyrene) was polymerized for 12 min (A) or overnight (B) at 22°C. Depolymerization was then induced by adding 30 μM latrunculin A. Data were fitted with a double-exponential equation, <i>I(t)</i> = <i>f</i>sexp(-t/τs) + (1 − <i>f</i>s)exp(-t/τf) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126262#pone.0126262.ref017" target="_blank">17</a>]. The parameters <i>f</i>s and τs respectively give the fraction and depolymerization rate of the slow-depolymerization population, and τf gives the depolymerization rate of the fast-depolymerization population. Inset shows the raw data before normalization, including those for the polymerization phase before addition of latrunculin A. (C) Trace of polymerization before addition of latrunculin A. Error bars indicate standard deviation (n = 3). (D) The critical concentrations of G146V and WT actins were measured by pelleting assay. Actins of 0.5~3.0 μM were polymerized for 3 hr, and were ultracentrifuged. Amounts of protein in resultant pellets were determined by Advanced Protein Assay Reagent. Error bars indicate standard deviation (n = 3).</p

Intramolecular FRET measurement system.

<p>(A) Donor and acceptor fluorophores were introduced at the tips of the two major domains of WT actin and G146V actin (FRET actin). Actin monomer (PDB ID: 1c0f) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126262#pone.0126262.ref048" target="_blank">48</a>] and filament (PDB ID: 3g37) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126262#pone.0126262.ref041" target="_blank">41</a>] were drawn by using UCSF Chimera software. (B) FRET actins were copolymerized with excess amounts of WT or G146V actin, 0.9% of which was biotinylated, and they were immobilized on a glass surface by using the biotin—avidin system. (C and D) A typical series of time-lapse fluorescence images and fluorescence intensities of donor and acceptor from a single actin subunit in a filament in the absence of myosin. During FRET, fluorescence of the acceptor (red) was higher than that of the background, while that of the donor (green) was lower. Upon photobleaching of the acceptor (red arrow), the fluorescence intensity of the donor increased. Subsequently, the donor was also bleached (green arrow).</p