Single Molecular Observation of Self-Regulated Kinesin Motility

Kinesin-1 is an ATP-driven molecular motor that transports various cargoes in cells, a process that can be regulated by the kinesin tail domain. Here, kinesin ATPase activity and motility were inhibited <i>in vitro</i> by interacting the kinesin heavy chain C-terminal tail domain with the kinesin N-terminal motor domain. Though the tail domain can directly interact with microtubules, we found 70% of tail domains failed to bind in the presence of >100 mM (high) KCl, which also modulated the ATPase inhibition manner. These observations suggest that self-inhibition of kinesin depends on electrostatic interactions between the motor domain, the tail domain, and a microtubule. Furthermore, we observed self-regulated behavior of kinesin at the single molecule level. The tail domain did not affect motility velocity, but it did lower the binding affinity of the motor domain to the microtubule. The decrement in binding was coupled to ATPase inhibition. Meanwhile, the tail domain transfected into living cells not only failed to bind to microtubules but also inhibited the motor domain and microtubule interaction, in agreement with our <i>in vitro</i> results. Furthermore, at high potassium concentrations, the self-regulation of kinesin observed in cells was like that <i>in vitro</i>. The results favor a way tail inhibition mechanism where the tail domain masks the microtubule binding site of the motor domain in high potassium concentration

Spontaneous Structural Changes in Actin Regulate G-F Transformation

<div><p>Transformations between G- (monomeric) and F-actin (polymeric) are important in cellular behaviors such as migration, cytokinesis, and morphing. In order to understand these transitions, we combined single-molecule Förster resonance energy transfer with total internal reflection fluorescence microscopy to examine conformational changes of individual actin protomers. We found that the protomers can take different conformational states and that the transition interval is in the range of hundreds of seconds. The distribution of these states was dependent on the environment, suggesting that actin undergoes spontaneous structural changes that accommodate itself to polymerization.</p> </div

Distribution of G-and F-actin Förster resonance energy transfer (FRET) efficiencies for all observed molecules.

<p>(A,B) In the case of G-actin, samples were fixed onto a glass surface via an anti-myc antibody. Two states can be seen (g and fg) (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045864#pone-0045864-g001" target="_blank">Fig. 1C–1E</a>). Increasing the ionic strength increased the fg state. (C) In the case of F-actin, another state (f state) appeared. Peak positions were determined by fitting FRET distributions for all observed forms of actin to a sum of 2 or 3 Gaussian distributions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045864#pone.0045864.s001" target="_blank">Figure S1</a>). Peak positions for the f, fg, and g states are 0.37, 0.54, and 0.75, respectively. The numbers of molecules represented in the histograms are 312, 109, and 374 in (a), (b), and (c), respectively.</p

Individual actin has several structures.

<p>(A) G-actin has 4 subdomains. Residues 41 (D-loop region in subdomain 2) and 374 (C-terminal region in subdomain 1) were labeled with Alexa dyes of different wavelengths. (B) Fluorescent images of the acceptor (left images) and donor (right images) captured simultaneously by an electron multiplying charged coupled device camera. (i) and (ii) indicate the donors and acceptors for different individual actin molecules, respectively. The corresponding Förster resonance energy transfer (FRET) efficiencies of (i) and (ii) are shown in (C) and (D), respectively. The scale bar is 5 µm. (C–E) Time series of the acceptor and donor intensities from different single G-actin molecules and the corresponding FRET efficiency histograms. Two states can be seen. (F) Time series of the acceptor and donor intensities from a single F-actin molecule and the corresponding FRET efficiency histogram. A state distinct from that observed in G-actin can be seen.</p

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

Model of actin structures based on the Förster resonance energy transfer (FRET) states.

<p>Using the FRET efficiencies, we surmise that the g and fg states in G-actin and F-actin are analogous. The f state appears only after polymerization and is, therefore, a specific state of F-actin.</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