Masahide Kikkawa

Switch-based mechanism of kinesin motors

Kinesin motors are specialized enzymes that use hydrolysis of ATP to generate force and movement along their cellular tracks, the microtubules. Although numerous biochemical and biophysical studies have accumulated much data that link microtubule-assisted ATP hydrolysis to kinesin motion, the structural view of kinesin movement remains unclear. This study of the monomeric kinesin motor KIF1A combines X-ray crystallography and cryo-electron microscopy, and allows analysis of force-generating conformational changes at atomic resolution. The motor is revealed in its two functionally critical states—complexed with ADP and with a non-hydrolysable analogue of ATP. The conformational change observed between the ADP-bound and the ATP-like structures of the KIF1A catalytic core is modular, extends to all kinesins and is similar to the conformational change used by myosin motors and G proteins. Docking of the ADP-bound and ATP-like crystallographic models of KIF1A into the corresponding cryo-electron microscopy maps suggests a rationale for the plus-end directional bias associated with the kinesin catalytic core.

Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids

Synaptotagmin acts as a Ca2+ sensor in neurotransmitter release through its two C2 domains. Ca2+-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca2+ binding to the C2B domain is more crucial for release than Ca2+ binding to the C2A domain. Here we show that Ca2+ induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C2B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C2B domain.

Kinesin Superfamily Protein 2A (KIF2A) Functions in Suppression of Collateral Branch Extension

Through interactions with microtubules, the kinesin superfamily of proteins (KIFs) could have multiple roles in neuronal function and development. During neuronal development, postmitotic neurons develop primary axons extending toward targets, while other collateral branches remain short. Although the process of collateral branching is important for correct wiring of the brain, the mechanisms involved are not well understood. In this study, we analyzed kif2a(-/-) mice, whose brains showed multiple phenotypes, including aberrant axonal branching due to overextension of collateral branches. In kif2a(-/-) growth cones, microtubule-depolymerizing activity decreased. Moreover, many individual microtubules showed abnormal behavior at the kif2a(-/-) cell edge. Based on these results, we propose that KIF2A regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules and that it plays an important role in the suppression of collateral branch extension.

High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations

Kinesin is an ATP‐driven microtubule (MT)‐based motor fundamental to organelle transport. Although a number of kinesin crystal structures have been solved, the structural evidence for coupling between the bound nucleotide and the conformation of kinesin is elusive. In addition, the structural basis of the MT‐induced ATPase activity of kinesin is not clear because of the absence of the MT in the structure. Here, we report cryo‐electron microscopy structures of the monomeric kinesin KIF1A–MT complex in two nucleotide states at about 10 Å resolution, sufficient to reveal the secondary structure. These high‐resolution maps visualized clear structural changes that suggest a mechanical pathway from the nucleotide to the neck linker via the motor core rotation. In addition, new nucleotide binding pocket conformations are observed that are different from X‐ray crystallographic structures; it is closed in the 5′‐adenylyl‐imidodiphosphate state, but open in the ADP state. These results suggest a structural model of biased diffusion movement of monomeric kinesin motor.

Dynein and kinesin share an overlapping microtubule‐binding site

Dyneins and kinesins move in opposite directions on microtubules. The question of how the same‐track microtubules are able to support movement in two directions remains unanswered due to the absence of details on dynein–microtubule interactions. To address this issue, we studied dynein–microtubule interactions using the tip of the microtubule‐binding stalk, the dynein stalk head (DSH), which directly interacts with microtubules upon receiving conformational change from the ATPase domain. Biochemical and cryo‐electron microscopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 Å, corresponding to the step size of dyneins. The DSH molecule was observed as a globular corn grain‐like shape that bound the same region as kinesin. Biochemical crosslinking experiments and image analyses of the DSH–kinesin head–microtubule complex revealed competition between DSH and the kinesin head for microtubule binding. Our results demonstrate that dynein and kinesin share an overlapping microtubule‐binding site, and imply that binding at this site has an essential role for these motor proteins.

Tau binding to microtubules does not directly affect microtubule-based vesicle motility

Tau protein is a major microtubule (MT)‐associated brain protein enriched in axons. Multiple functional roles are proposed for tau protein, including MT stabilization, generation of cell processes, and targeting of phosphotransferases to MTs. Recently, experiments involving exogenous tau expression in cultured cells suggested a role for tau as a regulator of kinesin‐1‐based motility. Tau was proposed to inhibit attachment of kinesin‐1 to MTs by competing for the kinesin‐1 binding site. In this work, we evaluated effects of tau on fast axonal transport (FAT) by using vesicle motility assays in isolated squid axoplasm. Effects of recombinant tau constructs on both kinesin‐1 and cytoplasmic dynein‐dependent FAT rates were evaluated by video microscopy. Exogenous tau binding to endogenous squid MTs was evidenced by a dramatic change in individual MT morphologies. However, perfusion of tau at concentrations ∼20‐fold higher than physiological levels showed no effect on FAT. In contrast, perfusion of a cytoplasmic dynein‐derived peptide that competes with kinesin‐1 and cytoplasmic dynein binding to MTs in vitro rapidly inhibited FAT in both directions. Taken together, our results indicate that binding of tau to MTs does not directly affect kinesin‐1‐ or cytoplasmic dynein‐based motilities. In contrast, our results provide further evidence indicating that the functional binding sites for kinesin‐1 and cytoplasmic dynein on MTs overlap.

Three-dimensional structures of the flagellar dynein–microtubule complex by cryoelectron microscopy

The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been observed. In this study, we establish a new method for reconstructing the 3D structure of the in vitro reconstituted ODA–microtubule complex and visualize nucleotide-dependent conformational changes using cryoelectron microscopy and image analysis. As the complex went from the rigor state to the relaxed state, the head domain of the β heavy chain shifted by 3.7 nm toward the B tubule and inclined 44° inwards. These observations suggest that there is a mechanism that converts head movement into the axonemal sliding motion.

The role of microtubules in processive kinesin movement

Kinesins are microtubule-based motors that are important for various intracellular transport processes. To understand the mechanism of kinesin movement, X-ray crystallography has been used to study the atomic structures of kinesin. However, as crystal structures of kinesin alone accumulate, it is becoming clear that kinesin structures should also be investigated with the microtubule to understand the contribution of the microtubule track to the nucleotide-induced conformational changes of kinesin. Recently, several high-resolution structures of kinesin with microtubules were obtained using cryo-electron microscopy. Comparison with X-ray crystallographic structures revealed the importance of the microtubule in determining the conformation of kinesin. Together with recent biophysical data, we describe different structural models of processive kinesin movement and provide a framework for future experiments.

Three-dimensional structure of cytoplasmic dynein bound to microtubules

Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dyneins head domain, formed by a ring-like arrangement of AAA+ domains, is located ≈280 Å away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dyneins microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dyneins power stroke.

A molecular ruler determines the repeat length in eukaryotic cilia and flagella

Existence of cellular structures with specific size raises a fundamental question in biology: How do cells measure length? One conceptual answer to this question is by a molecular ruler, but examples of such rulers in eukaryotes are lacking. In this work, we identified a molecular ruler in eukaryotic cilia and flagella. Using cryo-electron tomography, we found that FAP59 and FAP172 form a 96–nanometer (nm)–long complex in Chlamydomonas flagella and that the absence of the complex disrupted 96-nm repeats of axonemes. Furthermore, lengthening of the FAP59/172 complex by domain duplication resulted in extension of the repeats up to 128 nm, as well as duplication of specific axonemal components. Thus, the FAP59/172 complex is the molecular ruler that determines the 96-nm repeat length and arrangements of components in cilia and flagella. A protein complex controls the length and assembly of repeat structures in eukaryotic cilia and flagella. Molecular ruler rules cilia and flagella length Cilia and flagella contain within their ultrastructure repeating structures at regularly spaced intervals. How does the cell measure length with nanometer precision? Oda et al. identify a flagella protein complex in Chlamydomonas that appears to act as a sort of molecula ruler to define repeat length. Genetic changes that would change the length of this protein led to corresponding changes in the length of repeats within the resulting flagella. Science, this issue p. 857