Yamato Niitani

High-Speed Angle-Resolved Imaging of a Single Gold Nanorod with Microsecond Temporal Resolution and One-Degree Angle Precision

We developed two types of high-speed angle-resolved imaging methods for single gold nanorods (SAuNRs) using objective-type vertical illumination dark-field microscopy and a high-speed CMOS camera to achieve microsecond temporal and one-degree angle resolution. These methods are based on: (i) an intensity analysis of focused images of SAuNR split into two orthogonally polarized components and (ii) the analysis of defocused SAuNR images. We determined the angle precision (statistical error) and accuracy (systematic error) of the resultant SAuNR (80 nm × 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of ∼1° for the azimuthal angle at a 10 μs temporal resolution, the defocused image analysis showed a superior angle accuracy of ∼5°. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of ∼1°, by fitting with simulated images. By taking advantage of the defocused image methods full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F1-ATPase, was measured with 3.3 μs temporal resolution. The time constants of the pauses waiting for the elementary steps of the ATP hydrolysis reaction and the torque generated in the mechanical steps have been successfully estimated. The high-speed angle-resolved SAuNR imaging methods will be applicable to the monitoring of the fast conformational changes of many biological molecular machines.

CK2 activates kinesin via induction of a conformational change

Significance Kinesin regulation by autoinhibition has been extensively studied. However, it is known that cargo-bound kinesin motion can be altered by various signaling pathways. How are these kinesins regulated? Kinesin regulation by the signaling kinase casein kinase 2 (CK2) was previously reported to activate inactive kinesin at the level of the single motor head domain, but the mechanism was unknown. Here, using a multidisciplinary approach, we discover that kinesin inactivation involves a specific conformational change in the molecule’s neck linker, which controls microtubule affinity and is reversed by CK2. Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2’s dual ability to reverse this repositioning and to promote ADP release.

Strain-Dependent Regulation of the Kinesin-1's Catalytic Activity as Studied by Disulfide-Crosslinking of the Neck Linker

Kinesin is a dimeric motor protein that hydrolyzes ATP and moves along microtubules in a hand-over-hand manner. To walk by alternately moving two motor heads, the trailing head should detach from the microtubule prior to the leading head and the detached head should preferentially bind to the forward tubulin-binding site. To explain these mechanisms, we hypothesized that ATP hydrolysis reaction of kinesin motor domain can be regulated depending on the direction of the tension posed to the neck linker: backward strain posed to the neck linker suppresses ATP hydrolysis in the leading head and the forward strain posed to the neck linker suppresses ADP release at the trailing position. To test this hypothesis, we constrained the neck linker in the forward or backward extended conformation using disulfide-crosslinking between cysteine residues on the head and the neck linker, and examined these effects on the microtubule affinity and ADP release kinetics. Single molecule fluorescent observation of the GFP-fused monomeric kinesin showed that when the neck linker was constrained in a backward extended conformation, the dwell time on the microtubule in the presence of saturating ATP was increased by a factor of 15 compared to unconstrained condition. In contrast, stopped-flow measurement showed that when the neck linker was constrained in a forward extended conformation, ADP release rate after microtubule-binding was significantly decreased. These results support the idea that ATP hydrolysis cycle of kinesins motor domain can be differently regulated depending on the direction of the neck linker extension.