A neuropathy-associated kinesin KIF1A mutation hyper-stabilizes the motor-neck interaction during the ATPase cycle.

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the ß7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on ß7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.

Kinesin-1 mediates proper ER folding of the CaV1.2 channel and maintains mouse glucose homeostasis.

Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is a principal mechanism for systemic glucose homeostasis, of which regulatory mechanisms are still unclear. Here we show that kinesin molecular motor KIF5B is essential for GSIS through maintaining the voltage-gated calcium channel CaV1.2 levels, by facilitating an Hsp70-to-Hsp90 chaperone exchange to pass through the quality control in the endoplasmic reticulum (ER). Phenotypic analyses of KIF5B conditional knockout (cKO) mouse beta cells revealed significant abolishment of glucose-stimulated calcium transients, which altered the behaviors of insulin granules via abnormally stabilized cortical F-actin. KIF5B and Hsp90 colocalize to microdroplets on ER sheets, where CaV1.2 but not Kir6.2 is accumulated. In the absence of KIF5B, CaV1.2 fails to be transferred from Hsp70 to Hsp90 via STIP1, and is likely degraded via the proteasomal pathway. KIF5B and Hsc70 overexpression increased CaV1.2 expression via enhancing its chaperone binding. Thus, ER sheets may serve as the place of KIF5B- and Hsp90-dependent chaperone exchange, which predominantly facilitates CaV1.2 production in beta cells and properly enterprises GSIS against diabetes.

The two-step cargo recognition mechanism of heterotrimeric kinesin.

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

Kinesin Kif3b mutation reduces NMDAR subunit NR2A trafficking and causes schizophrenia-like phenotypes in mice.

The transport of N-methyl-d-aspartate receptors (NMDARs) is crucial for neuronal plasticity and synapse formation. Here, we show that KIF3B, a member of the kinesin superfamily proteins (KIFs), supports the transport of vesicles simultaneously containing NMDAR subunit 2A (NR2A) and the adenomatous polyposis coli (APC) complex. Kif3b+/- neurons exhibited a reduction in dendritic levels of both NR2A and NR2B due to the impaired transport of NR2A and increased degradation of NR2B. In Kif3b+/- hippocampal slices, electrophysiological NMDAR response was found decreased and synaptic plasticity was disrupted, which corresponded to a common feature of schizophrenia (SCZ). The histological features of Kif3b+/- mouse brain also mimicked SCZ features, and Kif3b+/- mice exhibited behavioral defects in prepulse inhibition (PPI), social interest, and cognitive flexibility. Indeed, a mutation of KIF3B was specifically identified in human SCZ patients, which was revealed to be functionally defective in a rescue experiment. Therefore, we propose that KIF3B transports NR2A/APC complex and that its dysfunction is responsible for SCZ pathogenesis.

KIF26A is an unconventional kinesin and regulates GDNF-Ret signaling in enteric neuronal development.

The kinesin superfamily proteins (KIFs) are motor proteins that transport organelles and protein complexes in a microtubule- and ATP-dependent manner. We identified KIF26A as a new member of the murine KIFs. KIF26A is a rather atypical member as it lacks ATPase activity. Mice with a homozygous deletion of Kif26a developed a megacolon with enteric nerve hyperplasia. Kif26a-/- enteric neurons showed hypersensitivity for GDNF-Ret signaling, and we find that KIF26A suppressed GDNF-Ret signaling by direct binding and inhibition of Grb2, an essential component of GDNF/Akt/ERK signaling. We therefore propose that the unconventional kinesin KIF26A plays a key role in enteric nervous system development by repressing a cell growth signaling pathway.

Kinesin superfamily motor proteins and intracellular transport.

Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different kinesins recognize and bind to specific cargos, as well as how kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of kinesin research.

The mechanisms of kinesin motor motility: lessons from the monomeric motor KIF1A.

Most kinesins move processively along microtubules by using energy derived from ATP hydrolysis. Almost all of the intermediate structures of this ATPase reaction cycle have been solved for the monomeric kinesin 3 family motor KIF1A. Based on this structural information, we propose a common mechanism of kinesin motility, focusing on the regulation of kinesin motility through their interaction with microtubules and by their 'neck-linker' region, which connects their motor domain to cargo and kinesin partner heads.

X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding.

The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.

ß-Tubulin mutations that cause severe neuropathies disrupt axonal transport.

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed ß-tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of ß-tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of ß-tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.

Characterizing KIF16B in neurons reveals a novel intramolecular "stalk inhibition" mechanism that regulates its capacity to potentiate the selective somatodendritic localization of early endosomes.

An organelle's subcellular localization is closely related to its function. Early endosomes require localization to somatodendritic regions in neurons to enable neuronal morphogenesis, polarized sorting, and signal transduction. However, it is not known how the somatodendritic localization of early endosomes is achieved. Here, we show that the kinesin superfamily protein 16B (KIF16B) is essential for the correct localization of early endosomes in mouse hippocampal neurons. Loss of KIF16B induced the aggregation of early endosomes and perturbed the trafficking and functioning of receptors, including the AMPA and NGF receptors. This defect was rescued by KIF16B, emphasizing the critical functional role of the protein in early endosome and receptor transport. Interestingly, in neurons expressing a KIF16B deletion mutant lacking the second and third coiled-coils of the stalk domain, the early endosomes were mistransported to the axons. Additionally, the binding of the motor domain of KIF16B to microtubules was inhibited by the second and third coiled-coils (inhibitory domain) in an ATP-dependent manner. This suggests that the intramolecular binding we find between the inhibitory domain and motor domain of KIF16B may serve as a switch to control the binding of the motor to microtubules, thereby regulating KIF16B activity. We propose that this novel autoregulatory "stalk inhibition" mechanism underlies the ability of KIF16B to potentiate the selective somatodendritic localization of early endosomes.

Defects in Synaptic Plasticity, Reduced NMDA-Receptor Transport, and Instability of Postsynaptic Density Proteins in Mice Lacking Microtubule-Associated Protein 1A.

Microtubule-associated protein 1A (MAP1A) is a member of the major non-motor microtubule-binding proteins. It has been suggested that MAP1A tethers NMDA receptors (NRs) to the cytoskeleton by binding with proteins postsynaptic density (PSD)-93 and PSD-95, although the function of MAP1A in vivo remains elusive. The present study demonstrates that mouse MAP1A plays an essential role in maintaining synaptic plasticity through an analysis of MAP1A knock-out mice. The mice exhibited learning disabilities, which correlated with decreased long-term potentiation and long-term depression in the hippocampal neurons, as well as a concomitant reduction in the extent of NR-dependent EPSCs. Surface expression of NR2A and NR2B subunits also decreased. Enhanced activity-dependent degradation of PSD-93 and reduced transport of NR2A/2B in dendrites was likely responsible for altered receptor function in neurons lacking MAP1A. These data suggest that tethering of NR2A/2B with the cytoskeleton through MAP1A is fundamental for synaptic function. SIGNIFICANCE STATEMENT: This work is the first report showing the significance of non-motor microtubule-associated protein in maintaining synaptic plasticity thorough a novel mechanism: anchoring of NMDA receptors to cytoskeleton supports transport of NMDA receptors and stabilizes postsynaptic density scaffolds binding to NMDA receptors. Newly generated mutant mice lacking MAP1A exhibited learning disabilities and reduced synaptic plasticity attributable to disruptions of the anchoring machinery.

Kinesin superfamily proteins (KIFs): Various functions and their relevance for important phenomena in life and diseases.

Kinesin superfamily proteins (KIFs) largely serve as molecular motors on the microtubule system and transport various cellular proteins, macromolecules, and organelles. These transports are fundamental to cellular logistics, and at times, they directly modulate signal transduction by altering the semantics of informational molecules. In this review, we will summarize recent approaches to the regulation of the transport destinations and to the physiological relevance of the role of these proteins in neuroscience, ciliary functions, and metabolic diseases. Understanding these burning questions will be essential in establishing a new paradigm of cellular functions and disease pathogenesis.

Phosphatidylinositol 4-phosphate 5-kinase alpha (PIPKα) regulates neuronal microtubule depolymerase kinesin, KIF2A and suppresses elongation of axon branches.

Neuronal morphology is regulated by cytoskeletons. Kinesin superfamily protein 2A (KIF2A) depolymerizes microtubules (MTs) at growth cones and regulates axon pathfinding. The factors controlling KIF2A in neurite development remain totally elusive. Here, using immunoprecipitation with an antibody specific to KIF2A, we identified phosphatidylinositol 4-phosphate 5-kinase alpha (PIPKα) as a candidate membrane protein that regulates the activity of KIF2A. Yeast two-hybrid and biochemical assays demonstrated direct binding between KIF2A and PIPKα. Partial colocalization of the clusters of punctate signals for these two molecules was detected by confocal microscopy and photoactivated localization microscopy. Additionally, the MT-depolymerizing activity of KIF2A was enhanced in the presence of PIPKα in vitro and in vivo. PIPKα suppressed the elongation of axon branches in a KIF2A-dependent manner, suggesting a unique PIPK-mediated mechanism controlling MT dynamics in neuronal development.

New simulated annealing approach considering helix bending applied to determine the 8.8Å structure of 15-protofilament microtubules.

The helix is an important motif in biological architectures. The helical structures of nanoscale proteins are principally determined by three-dimensional (3D) reconstruction from electron micrographs. However, bending or distortion of flexible helices and the low contrast of the images recorded by cryo-electron microscopy, prevent the analysis from reaching high resolution. We have developed a novel helical reconstruction method that overcomes these issues, and present the processing of microtubule images to demonstrate its application. Cropping long helical structures into small square pieces allows bending or distortion of the helices to be accounted for. The initial image-frames are automatically positioned assuming perfect helical symmetry. A simulated annealing (SA)-based algorithm is then used to adjust the framing. This is guided by the contrast of 2D averages, which serve as an accuracy index. After the initial 3D reconstruction, the position and orientation of each average image is iteratively adjusted to give the best match between the input average and the reprojection from the reconstruction. Finally, reconstructions from images recorded at different defocus values, are aligned and averaged to compensate the contrast transfer modulation and improve the resolution. The method successfully determined the structure of a 15-protofilament microtubule. The 8.8Å resolution (7.8Å using the 0.143 FSC criterion) attained allows differences between the α- and ß- tubulins to be discerned in the absence of a molecular landmark such as microtubule-associated proteins, for the first time by electron microscopy. The SA-based method is applicable to other helical protein complexes and in general to helical structures.

Kinesin-1/Hsc70-dependent mechanism of slow axonal transport and its relation to fast axonal transport.

Cytoplasmic protein transport in axons ('slow axonal transport') is essential for neuronal homeostasis, and involves Kinesin-1, the same motor for membranous organelle transport ('fast axonal transport'). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin-1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ-like domain of the kinesin light chain in the Kinesin-1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin-1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant-negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.

A novel in-frame deletion in KIF5C gene causes infantile onset epilepsy and psychomotor retardation.

Motor proteins, encoded by Kinesin superfamily (KIF) genes, are critical for brain development and plasticity. Increasing studies reported KIF's roles in neurodevelopmental disorders. Here, a 6 years and 3 months-old Chinese boy with markedly symptomatic epilepsy, intellectual disability, brain atrophy, and psychomotor retardation was investigated. His parents and younger sister were phenotypically normal and had no disease-related family history. Whole exome sequencing identified a novel heterozygous in-frame deletion (c.265_267delTCA) in exon 3 of the KIF5C in the proband, resulting in the removal of evolutionarily highly conserved p.Ser90, located in its ATP-binding domain. Sanger sequencing excluded the proband's parents and family members from harboring this variant. The activity of ATP hydrolysis in vitro was significantly reduced as predicted. Immunofluorescence studies showed wild-type KIF5C was widely distributed throughout the cytoplasm, while mutant KIF5C was colocalized with microtubules. The live-cell imaging of the cargo-trafficking assay revealed that mutant KIF5C lost the peroxisome-transporting ability. Drosophila models also confirmed p.Ser90del's essential role in nervous system development. This study emphasized the importance of the KIF5C gene in intracellular cargo-transport as well as germline variants that lead to neurodevelopmental disorders and might enable clinicians for timely and accurate diagnosis and disease management in the future.

Regulation of NMDA receptor transport: a KIF17-cargo binding/releasing underlies synaptic plasticity and memory in vivo.

Regulation of NMDA receptor trafficking is crucial to modulate neuronal communication. Ca(2+)/calmodulin-dependent protein kinase phosphorylates the tail domain of KIF17, a member of the kinesin superfamily, to control NMDA receptor subunit 2B (GluN2B) transport by changing the KIF17-cargo interaction in vitro. However, the mechanisms of regulation of GluN2B transport in vivo and its physiological significance are unknown. We generated transgenic mice carrying wild-type KIF17 (TgS), or KIF17 with S1029A (TgA) or S1029D (TgD) phosphomimic mutations in kif17(-/-) background. TgA/kif17(-/-) and TgD/kif17(-/-) mice exhibited reductions in synaptic NMDA receptors because of their inability to load/unload GluN2B onto/from KIF17, leading to impaired neuronal plasticity, CREB activation, and spatial memory. Expression of GFP-KIF17 in TgS/kif17(-/-) mouse neurons rescued the synaptic and behavioral defects of kif17(-/-) mice. These results suggest that phosphorylation-based regulation of NMDA receptor transport is critical for learning and memory in vivo.

Nodal flow transfers polycystin to determine mouse left-right asymmetry.

Left-dominant [Ca2+]i elevation on the left margin of the ventral node furnishes the initial laterality of mouse embryos. It depends on extracellular leftward fluid flow (nodal flow), fibroblast growth factor receptor (FGFR)/sonic hedgehog (Shh) signaling, and the PKD1L1 polycystin subunit, of which interrelationship is still elusive. Here, we show that leftward nodal flow directs PKD1L1-containing fibrous strands and facilitates Nodal-mediated [Ca2+]i elevation on the left margin. We generate KikGR-PKD1L1 knockin mice in order to monitor protein dynamics with a photoconvertible fluorescence protein tag. By imaging those embryos, we have identified fragile meshwork being gradually transferred leftward involving pleiomorphic extracellular events. A portion of the meshwork finally bridges over the left nodal crown cells in an FGFR/Shh-dependent manner. As PKD1L1 N-term is predominantly associated with Nodal on the left margin and that PKD1L1/PKD2 overexpression significantly augments cellular Nodal sensitivity, we propose that leftward transfer of polycystin-containing fibrous strands determines left-right asymmetry in developing embryos.

KIF4 regulates neuronal morphology and seizure susceptibility via the PARP1 signaling pathway.

Epilepsy is a common neurological disease worldwide, and one of its causes is genetic abnormalities. Here, we identified a point mutation in KIF4A, a member of kinesin superfamily molecular motors, in patients with neurological disorders such as epilepsy, developmental delay, and intellectual disability. KIF4 is involved in the poly (ADP-ribose) polymerase (PARP) signaling pathway, and the mutation (R728Q) strengthened its affinity with PARP1 through elongation of the KIF4 coiled-coil domain. Behavioral tests showed that KIF4-mutant mice exhibited mild developmental delay with lower seizure threshold. Further experiments revealed that the KIF4 mutation caused aberrant morphology in dendrites and spines of hippocampal pyramidal neurons through PARP1-TrkB-KCC2 pathway. Furthermore, supplementing NAD, which activates PARP1, could modulate the TrkB-KCC2 pathway and rescue the seizure susceptibility phenotype of the mutant mice. Therefore, these findings indicate that KIF4 is engaged in a fundamental mechanism regulating seizure susceptibility and could be a potential target for epilepsy treatment.

The KIF3 motor transports N-cadherin and organizes the developing neuroepithelium.

In the developing brain, the organization of the neuroepithelium is maintained by a critical balance between proliferation and cell-cell adhesion of neural progenitor cells. The molecular mechanisms that underlie this are still largely unknown. Here, through analysis of a conditional knockout mouse for the Kap3 gene, we show that post-Golgi transport of N-cadherin by the KIF3 molecular motor complex is crucial for maintaining this balance. N-cadherin and beta-catenin associate with the KIF3 complex by co-immunoprecipitation, and colocalize with KIF3 in cells. Furthermore, in KAP3-deficient cells, the subcellular localization of N-cadherin was disrupted. Taken together, these results suggest a potential tumour-suppressing activity for this molecular motor.