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.

In this paper (Cell 95[6], 829-837), we described the direction of the nodal cilia rotation as counterclockwise (Figure 6B). However, our recent analyses with higher spatiotemporal resolution revealed that the actual direction is clockwise when seen from the ventral side (above the nodal pit cells). In our previous observation with lower temporal resolution (10 frames per second), the direction of the rapid rotation ‫01ف(‬ rounds per second) of the nodal cilia was misinterpreted due to the artifact caused by the strobe effect. It should be noted that this artifact only affects the apparent direction of the rotary movement. Thus, the direction of the linear movement of the beads in the extraembryonic fluid or the direction of the nodal flow is leftward albeit the low temporal resolution of our previous analysis. Therefore, the clockwise rotation of the nodal cilia causes the leftward nodal flow. For further details, refer to our recent paper Y. Okada et al. (Molecular Cell, 1999, in press).

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 exogenous 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.

In cells, molecular motors operate in polarized sorting of molecules, although the steering mechanisms of motors remain elusive. In neurons, the kinesin motor conducts vesicular transport such as the transport of synaptic vesicle components to axons and of neurotransmitter receptors to dendrites, indicating that vesicles may have to drive the motor for the direction to be correct. Here we show that an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor subunit--GluR2-interacting protein (GRIP1)--can directly interact and steer kinesin heavy chains to dendrites as a motor for AMPA receptors. As would be expected if this complex is functional, both gene targeting and dominant negative experiments of heavy chains of mouse kinesin showed abnormal localization of GRIP1. Moreover, expression of the kinesin-binding domain of GRIP1 resulted in accumulation of the endogenous kinesin predominantly in the somatodendritic area. This pattern was different from that generated by the overexpression of the kinesin-binding scaffold protein JSAP1 (JNK/SAPK-associated protein-1, also known as Mapk8ip3), which occurred predominantly in the somatoaxon area. These results indicate that directly binding proteins can determine the traffic direction of a motor protein.

Neurons develop a highly polarized morphology consisting of dendrites and a long axon. Both axons and dendrites contain microtubules and microtubule-associated proteins (MAPs) with characteristic structures. Among MAPs, MAP2 is specifically expressed in dendrites whereas MAP2C and tau are abundant in the axon. But the influence of MAP2, MAP2C and tau on the organization of microtubule domains in dendrites versus axons is unknown. Both MAP2 and tau induce microtubule bundle formation in fibroblasts after transfection of complementary DNAs, and a long process resembling an axon is extended in Sf9 cells infected with recombinant baculovirus expressing tau. We have now expressed MAP2 and MAP2C in Sf9 cells in order to compare their morphology and the arrangement of their microtubules to that found in Sf9 cells expressing tau. We report here that the spacing between microtubules depends on the MAP expressed: in cells expressing MAP2, the distance is similar to that found in dendrites, whereas the spacing between microtubules in cells expressing MAP2C or tau is similar to that found in axons.