Michio Tomishige

Role of Phosphatidylinositol(4,5)bisphosphate Organization in Membrane Transport by the Unc104 Kinesin Motor

Unc104 (KIF1A) kinesin transports membrane vesicles along microtubules in lower and higher eukaryotes. Using an in vitro motility assay, we show that Unc104 uses a lipid binding pleckstrin homology (PH) domain to dock onto membrane cargo. Through its PH domain, Unc104 can transport phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2)-containing liposomes with similar properties to native vesicles. Interestingly, liposome movement by monomeric Unc104 motors shows a very steep dependence on PtdIns(4,5)P2 concentration (Hill coefficient of approximately 20), even though liposome binding is noncooperative. This switch-like transition for movement can be shifted to lower PtdIns(4,5)P2 concentrations by the addition of cholesterol/sphingomyelin or GM1 ganglioside/cholera toxin, conditions that produce raft-like behavior of Unc104 bound to lipid bilayers. These studies suggest that clustering of Unc104 in PtdIns(4,5)P2-containing rafts provides a trigger for membrane transport.

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.

Application of laser tweezers to studies of the fences and tethers of the membrane skeleton that regulate the movements of plasma membrane proteins.

Publisher Summary This chapter discusses the application of laser tweezers to studies of the fences and tethers of the membrane skeleton that regulate the movements of plasma membrane proteins. The cellular mechanisms that control the movement and assembly of membrane proteins in the plasma membrane are currently undergoing rapid evolution. The chapter illustrates the application of laser tweezers to studies of the mechanisms by which cells regulate the movement and lateral diffusion of membrane proteins. In these experiments, gold or latex particles are attached to membrane proteins, and the particle-membrane protein complex is captured by laser tweezers and moved laterally along the plasma membrane by scanning the trapping laser beam. Two types of major interactions between the membrane skeleton and membrane proteins have been found: One is the binding of the membrane proteins to the membrane skeleton, and the other is the corralling or fence effects of the membrane skeleton on the lateral diffusion of membrane proteins.

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.

Activation of mitotic kinesin by microtubule bundling

Kinesin-5 family members cross-link and slide parallel microtubules of opposite polarity, an activity that is essential for the formation of a bipolar spindle during mitosis. In this issue, Kapitein et al. (Kapitein, L.C., B.H. Kwok, J.S. Weinger, C.F. Schmidt, T.M. Kapoor, and E.J.G. Peterman. 2008. J. Cell Biol. 182:421–428) demonstrate that microtubule cross-linking triggers the conversion of kinesin-5 motility from a diffusive mode to a directional mode, initiating antiparallel microtubule sliding.

Single molecule FRET observation of kinesin-1's head-tail interaction on microtubule

Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility.

Single-molecule fluorescence imaging of kinesin using linear zero-mode waveguides

For the analysis of kinesin motility, we demonstrate an observation system to simultaneously visualize kinesin and ATP molecules at the single molecule level using Linear Zero-Mode Waveguides (LZMWs). LZMWs allow fluorescent imaging at up to micromolar concentration by confining the excitation volume, which is high enough for studying enzymatic reactions such as ATP hydrolysis. LZMWs were fabricated by electron-beam lithography and lift-off process. Moreover, we developed a method to immobilize microtubules in LZMWs to observe kinesin motility.

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.

Forward Stepping Mechanism of Kinesin-1 Studied using Asymmetrically-Joined Two-Headed Monomer

Kinesin-1 moves processively along microtubule by alternately moving two motor domains, but the mechanism of the preferential forward stepping is still controversial. The “neck linker-docking model” proposes that the neck linker docking of the microtubule-bound head generates forward bias of the tethered head. However, our recent structural analysis of kinesin dimer (Makino et al.) suggested an alternate model in which the tethered head position does not necessarily be biased because the tethered head is not allowed to bind to the rear tubulin-binding site due to a steric constraint on its neck linker and can only release ADP at the forward binding site (“biased-binding model”). To distinguish these mechanisms as alternate steps, we engineered two-headed monomer kinesin by joining two motor heads in tandem on a single polypeptide, in which the neck linker of first head (N-head) is connected to second head (C-head) so that it can propel C-head forward, whereas the neck linker of C-head is free. Single molecule fluorescence observation showed that this two-headed monomer moves processively along microtubules although the velocity was smaller than wild-type dimer by four-fold. In addition, FIONA measurement of individual head showed that both heads takes discrete 16 nm steps, illustrating that this monomer moves by alternately exchanging two heads. Then we measured the dwell time of alternate steps using single molecule FRET and found that forward-stepping of C-head presumably driven by the neck linker docking was less efficient than the forward-stepping of N-head, because the tethered C-head often rebinds to the rear-binding site. These results suggest that biased-binding mechanism is more efficient to drive forward stepping, because rebinding of the tethered head to the rear-binding site is effectively prohibited.