Teppei Mori

How kinesin waits between steps

Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules by hydrolysing ATP and moving processively in 8-nm steps. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains (‘heads’), a process driven by a conformational change in the neck-linker domain of kinesin. However, the ‘waiting conformation’ of kinesin between steps remains controversial—some models propose that kinesin adopts a one-head-bound intermediate, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.

Hierarchical transport of nanoparticles in a lyotropic lamellar phase

The dynamics of nanosized colloidal particles dispersed in a hyper-swollen lyotropic lamellar phase of a nonionic surfactant has been studied by ac electrophoretic light scattering and direct tracking of particles under a microscope. The frequency spectrum of electrophoretic mobility shows two relaxation processes. These are originated from the hindrance of free diffusion of particles by the interaction between membranes and particles. By direct tracking measurement, we find that particles jump from site to site where they stay for a long time. This trap-jump process greatly decreases the mobility at low frequencies.

Optimal Size of the Neck Linker is Important for the Coordinated Processive Movement of Kinesin-1

Kinesin-1 is a dimeric motor protein that walks along microtubules by alternately moving two motor ‘heads’. Several recently published papers including ours provided evidences that kinesin dimer takes one-head-bound state while waiting for ATP and ATP-binding triggers the tethered head to bind to the forward tubulin-binding site. However, it is still not clear why rebinding of the tethered head, which is freely diffusing, to microtubule is prohibited during the ATP-waiting state. To explain this mechanism, we proposed a model based on the crystal structural analysis (Makino et al, this meeting) that ADP release of the tethered head is prohibited because the neck linker would be stretched out if both heads become nucleotide-free due to a steric hindrance posed on the neck linker. This model predicts that if the neck linker is artificially extended, the tethered head can easily rebind to the microtubule. To test this prediction, we engineered neck linker extended mutants by inserting poly-Gly residues and observed their conformational states using single-molecule FRET technique. We found that 5 amino acid extension of the neck linker allows the tethered head to rapidly rebind to the microtubule even in the absence of ATP, and that in this state both neck linkers adopt backward-pointing conformation. The neck linker extended mutants showed processive motility with reduced velocities compare to the wild-type, although the microtubule-activated ATPase rate was not changed, which are consistent with our previous results using poly-Pro insertion (Yildiz et al 2008). There results suggest that optimal size of the neck linker is important to prevent rebinding of the tethered head while waiting for ATP and to efficiently couple ATP hydrolysis energy with forward step.

Hierarchical Dynamics of Nano-Particles in Lyotropic Lamellar Phase

ABSTRACT Dynamics of nano-sized colloidal particles dispersed in a dilute lyotropic lamellar phase of a nonionic surfactant has been studied experimentally by ac electrophoretic light scattering and direct tracking under a microscope. The obtained frequency spectrum of complex electrophoretic mobility shows two relaxation processes at about 1 kHz (HF relaxation) and a few Hz (LF relaxation). These relaxations are due to the hindrance of free diffusion of particles by the hierarchical local static and dynamical structures of lamellar phase. From the direct tracking of fluorescent-labeled particles under a microscope, we find that particles show jump from sites to sites where they stay for a long time. This trap-jump process extremely decreases their mobility at low frequencies.