A Change in the Radius of Rotation of F1-ATPase Indicates a Tilting Motion of the Central Shaft

AbstractF1-ATPase is a water-soluble portion of FoF1-ATP synthase and rotary molecular motor that exhibits reversibility in chemical reactions. The rotational motion of the shaft subunit γ has been carefully scrutinized in previous studies, but a tilting motion of the shaft has never been explicitly postulated. Here we found a change in the radius of rotation of the probe attached to the shaft subunit γ between two different intermediate states in ATP hydrolysis: one waiting for ATP binding, and the other waiting for ATP hydrolysis and/or subsequent product release. Analysis of this radial difference indicates a ∼4° outward tilting of the γ-subunit induced by ATP binding. The tilt angle is a new parameter, to our knowledge, representing the motion of the γ-subunit and provides a new constraint condition of the ATP-waiting conformation of F1-ATPase, which has not been determined as an atomic structure from x-ray crystallography

Motor generated torque drives coupled yawing and orbital rotations of kinesin coated gold nanorods

Kinesin motor domains generate impulses of force and movement that have both translational and rotational (torque) components. Here, we ask how the torque component influences function in cargo-attached teams of weakly processive kinesins. Using an assay in which kinesin-coated gold nanorods (kinesin-GNRs) translocate on suspended microtubules, we show that for both single-headed KIF1A and dimeric ZEN-4, the intensities of polarized light scattered by the kinesin-GNRs in two orthogonal directions periodically oscillate as the GNRs crawl towards microtubule plus ends, indicating that translocating kinesin-GNRs unidirectionally rotate about their short (yaw) axes whilst following an overall left-handed helical orbit around the microtubule axis. For orientations of the GNR that generate a signal, the period of this short axis rotation corresponds to two periods of the overall helical trajectory. Torque force thus drives both rolling and yawing of near-spherical cargoes carrying rigidly-attached weakly processive kinesins, with possible relevance to intracellular transport

CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules

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

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