Prof. Dr. Nobutaka Hirokawa

Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo

Molecular Motors' Kinesin Superfamily Proteins (KIFs)' Key Molecules for Neuronal Function: From Gene, Structure, Dynamics to Function and Disease

Cells transport and sort various proteins and lipids following synthesis as distinct kinds of membranous organelles and protein complexes to the correct destinations at appropriate velocities. This is true for all kinds of cells, both nonpolarized cells such as fibroblasts and polarized cells such as neurons and epithelial cells.

A typical polarized cell, neuron is composed of dendrites, a cell body and a long axon along the direction of impulse propagation. Because of the lack of protein synthesis machinery in the axon, all the proteins required in the axon and synaptic terminal have to be transported down the axon following synthesis in the cell body. Because similar mechanisms are observed in all other cells, the neuron serves as a good model system to study the general mechanisms of organelle transport.In this sense organelle transport is fundamentally important for cellular morphogenesis and functioning. To elucidate this mechanism we have identified and characterized more than 20 new kinesin superfamily proteins, KIFs.

In this symposium I will focus on KIF1A, KIF3, KIF 17 and KIF 1B beta and introduce our recent data.

A single conventional kinesin (dimeric) molecule can move "processively" along a microtubule for more than 1 micron m before detaching from it. The prevailing explanation for this movement is the " walking model", which envisions that each of two motor domains (heads) of the kinesin molecule binds coordinatly to the microtubule. This implies that each kinesin molecule must have two heads to "walk" and that a single-headed kinesin could not move processively. We identified a monomeric motor KIF1A which transports precursors of synaptic vesicles and play essential role on neuronal function and neuronal survival. A motor-domain construct of KIF1A, a single-headed kinesin superfamily protein was shown to move processively along the microtubule for more than 1 micron m. The movement along the microtubules was stochastic and fitted a biased Brownian-movement model. Further, cryoEM study revealed that polylysine loop (k-loop) in loop 12 is unique for monomeric KIF1A and play an important role for the processivity. Our recent X-ray crystallography study combined with cryo-EM revealed how KIF 1A change conformation and move along microtubules.

KIF3 is a heterotrimeric motor composed of KIF3A and KIF3B heterodimer with an associated protein KAP3 and is expressed abundantly in the nervous system while it is expressed in other type of cells ubiquitously. To analyse the function of KIF3 we disrupted kif3A and kif3B respectively 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 randamized in the heart loop and the direction of embryonic turning. It is known that there are a gene cascade expressed exclusively at the left side of the embryo and important for the left-right determination such as lefty 2, lefty1 and nodal. In the mutants the expression of lefty-2, the most upstream gene was either bilateral or absent. Furthermore, the epithelial cells in the node, which is thought to be essential for L-R determination, lacked monocilia. Immunocytochemistry revealed KIF3A and KIF3B localization in wild type nodal cilia. Because this monocilium is composed of 9+0 microtubules it is thought to be immotile. However, we found by video microscopy that these cilia were motile and rotating and generated a leftward flow of extraembryonic fluid at the node region named "nodal flow". These data suggest that KIF3 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. Thus, motor proteins are involved also in the key phenomenon controlling important developmental events.

KIF 17 is a neuron-specific new molecular motor which is dominantly localized in nerve dendrites and performs selective transport of NMDA receptor containing vesicles along microtubules in dendrites. NMDA receptors are known to be fundamental for learning and memory. The selective transport is accomplished by direct interaction of the KIF 17 tail with a PDZ domain of mLin-10(Mint1), which is a constituent of a large protein complex including mLin-2(CASK), mLin-7(MALS/Velis) and NMDA receptor subunit NR2B. This interaction, specific for a neurotransmitter receptor critically important for plasticity in the postsynaptic terminal, may be a regulatory point at synaptic plasticity and neuronal morphogenesis.

KIF 1B beta is a novel isoform of KIF 1B alpha which transports mitochondria. KIF 1B beta is distinct from KIF 1B alpha in its cargo-binding domain. KIF 1B knockout mice die at birth from apnea due to nervous system defects. Death of knockout neurons in culture can be rescued by expression of the beta isoform. The KIF 1B heterozygotes have a defect in transporting synaptic vesicle precursors and suffer from progressive muscle weakness similar to human neuropathies. Charcot-Marie-Tooth disease type 2A was mapped previously to an interval containing KIF 1B. We show that CMT2A patients contain a loss of function mutation in the motor domain of the KIF 1B gene. This is the first indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathy.

Thus molecular motors play not only very fundamental roles on neuronal functions but also significant roles on key events in the development such as Left-Right determination. Furthermore, they become a cause of human disease.