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The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.

SummaryThe leftward flow in extraembryonic fluid is critical for the initial determination of the left-right axis of mouse embryos. It is unclear if this is a conserved mechanism among other vertebrates and how the directionality of the flow arises from the motion of cilia. In this paper, we show that rabbit and medakafish embryos also exhibit a leftward fluid flow in their ventral nodes. In all cases, primary monocilia present a clockwise rotational-like motion. Observations of defective ciliary dynamics in mutant mouse embryos support the idea that the posterior tilt of the cilia during rotational-like beating can explain the leftward fluid flow. Moreover, we show that this leftward flow may produce asymmetric distribution of exogenously introduced proteins, suggesting morphogen gradients as a subsequent mechanism of left-right axis determination. Finally, we experimentally and theoretically characterize under which conditions a morphogen gradient can arise from the flow.

A two-headed structure has been widely believed to be essential for the kinesin molecular motor to move processively on the track, microtubules. However, we have recently demonstrated that a monomeric motor domain construct of KIF1A (C351), a kinesin superfamily protein, moves processively, taking about 700 steps before being detached from microtubules. To elucidate the mechanism of its single-headed processivity, we examined the C351-MT interaction by mutant analysis and high-resolution cryo-EM. Mutant analysis indicated the importance of a highly positively charged loop, the "K loop," for such processivity. A 15 A resolution structure unambiguously docked with the available atomic models revealed "K loop" as an extra microtubule-binding domain specific to KIF1A, and bound to the C terminus of tubulin. The site-specific cross-linking further confirmed this model.

To further elucidate the mechanism of organelle transport, we cloned a novel member of the mouse kinesin superfamily, KIF1B. This N-terminal-type motor protein is expressed ubiquitously in various kinds of tissues. In situ hybridization revealed that KIF1B is expressed abundantly in differentiated nerve cells. Interestingly, KIF1B works as a monomer, having a microtubule plus end-directed motility. Our rotary shadowing electron microscopy revealed mostly single globular structures. Immunocytochemically, KIF1B was colocalized with mitochondria in vivo. Furthermore, a subcellular fractionation study showed that KIF1B was concentrated in the mitochondrial fraction, and purified KIF1B could transport mitochondria along microtubules in vitro. These data strongly suggested that KIF1B works as a monomeric motor for anterograde transport of mitochondria.

Unlike other kinesins, middle motor domain-type kinesins depolymerize the microtubule from its ends. To elucidate its mechanism, we solved the X-ray crystallographic structure of KIF2C, a murine member of this family. Three major class-specific features were identified. The class-specific N-terminal neck adopts a long and rigid helical structure extending out vertically into the interprotofilament groove. This structure explains its dual roles in targeting to the end of the microtubule and in destabilization of the lateral interaction of the protofilament. The loop L2 forms a unique finger-like structure, long and rigid enough to reach the next tubulin subunit to stabilize the peeling of the protofilament. The open conformation of the switch I loop could be reversed by the shift of the microtubule binding L8 loop, suggesting its role as the sensor to trigger ATP hydrolysis. Mutational analysis supports these structural implications.

Mouse kif5B gene was disrupted by homologous recombination. kif5B −/− mice were embryonic lethal with a severe growth retardation at 9.5–11.5 days postcoitum. To analyze the significance of this conventional kinesin heavy chain in organelle transport, we studied the distribution of major organelles in the extraembryonic cells. The null mutant cells impaired lysosomal dispersion, while brefeldin A could normally induce the breakdown of their Golgi apparatus. More prominently, their mitochondria abnormally clustered in the perinuclear region. This mitochondrial phenotype was reversed by an exogeneous expression of KIF5B, and a subcellular fractionation revealed that KIF5B is associated with mitochondria. These data collectively indicate that kinesin is essential for mitochondrial and lysosomal dispersion rather than for the Golgi-to-ER traffic in these cells.

Microtubule-dependent motor, murine KIF3B, was disrupted by gene targeting. The null mutants did not survive beyond midgestation, exhibiting growth retardation, pericardial sac ballooning, and neural tube disorganization. Prominently, the left–right asymmetry was randomized in the heart loop and the direction of embryonic turning. lefty-2 expression was either bilateral or absent. Furthermore, the node lacked monocilia while the basal bodies were present. Immunocytochemistry revealed KIF3B localization in wild-type nodal cilia. Video microscopy showed that these cilia were motile and generated a leftward flow. These data suggest that KIF3B is essential for the left–right determination through intraciliary transportation of materials for ciliogenesis of motile primary cilia that could produce a gradient of putative morphogen along the left–right axis in the node.