The kinesin, dynein, and myosin superfamily molecular motors have fundamental roles in neuronal function, plasticity, morphogenesis, and survival by transporting cargos such as synaptic vesicle ...precursors, neurotransmitter and neurotrophic factor receptors, and mRNAs within axons, dendrites, and synapses. Recent studies have begun to clarify the mechanisms of cargo selection and directional transport in subcellular compartments. Furthermore, molecular genetics has revealed unexpected roles for molecular motors in brain wiring, neuronal survival, neuronal plasticity, higher brain function, and control of central nervous system and peripheral nervous system development. Finally, it is also evident that molecular motors are critically involved in neuronal disease pathogenesis. Thus, molecular motor research is becoming an exciting frontier of neuroscience.
Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde ...and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.
Dynein is a microtubule‐dependent motor protein required for cell division, retrograde intracellular transport, and intraflagellar transport (IFT). Dynein 1 and dynein 2 serve as molecular motors in ...the cytoplasm and cilia, respectively. Each dynein consists of multiple subunits. Although the components of dynein 1 and dynein 2 are different and specific in most species, a previous study has suggested that dynein intermediate chain subunit DYCI‐1 is shared by both dynein 1 and 2 in Caenorhabditis elegans (C. elegans). Here, we show that C. elegans has two dynein intermediate chains—DYCI‐1 and WDR‐60—and their functions are different. Mutational analysis showed that dyci‐1 is essential for the retrograde axonal transport of synaptic vesicles. In the same mutant allele, IFT is not affected at all. Instead, wdr‐60 is essential for IFT. Thus, we suggest that dynein 1 and dynein 2 have specific intermediate chains in C. elegans as in other organisms.
Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motors that serve as sources of force for intracellular transport and cell division. Recent studies have revealed new roles of ...KIFs as microtubule stabilizers and depolymerizers, and these activities are fundamental to cellular morphogenesis and mammalian development. KIF2A and KIF19A have microtubule-depolymerizing activities and regulate the neuronal morphology and cilia length, respectively. KIF21A and KIF26A work as microtubule stabilizers that regulate axonal morphology. Morphological defects that are similar to human diseases are observed in mice in which these KIF genes have been deleted. Actually, KIF2A and KIF21A have been identified as causes of human neuronal diseases. In this review, the functions of these atypical KIFs that regulate microtubule dynamics are discussed. Moreover, some interesting unanswered questions and hypothetical answers to them are discussed.
KIF5A is a kinesin superfamily motor protein that transports various cargos in neurons. Mutations in Kif5a cause familial amyotrophic lateral sclerosis (ALS). These ALS mutations are in the intron of ...Kif5a and induce mis‐splicing of KIF5A mRNA, leading to splicing out of exon 27, which in human KIF5A encodes the cargo‐binding tail domain of KIF5A. Therefore, it has been suggested that ALS is caused by loss of function of KIF5A. However, the precise mechanisms regarding how mutations in KIF5A cause ALS remain unclear. Here, we show that an ALS‐associated mutant of KIF5A, KIF5A(Δexon27), is predisposed to form oligomers and aggregates in cultured mouse cell lines. Interestingly, purified KIF5A(Δexon27) oligomers showed more active movement on microtubules than wild‐type KIF5A in vitro. Purified KIF5A(∆exon27) was prone to form aggregates in vitro. Moreover, KIF5A(Δexon27)‐expressing Caenorhabditis elegans neurons showed morphological defects. These data collectively suggest that ALS‐associated mutations of KIF5A are toxic gain‐of‐function mutations rather than simple loss‐of‐function mutations.
ALS‐associated KIF5A protein aggregates in cells and in vitro and has neurotoxicity.
The development and functions of neurons are supported by axonal transport. Axonal transport is a complex process whose regulation involves multiple molecules, such as microtubules, ...microtubule-associated proteins, kinases, molecular motors, and motor binding proteins. Gain of function and loss of function mutations of genes that encode these proteins often lead to human axonal neuropathy. Caenorhabditis elegans provides a powerful genetic system to study the consequences of gene mutations for axonal transport. Here, we discuss advantages and limitations of using C. elegans, propose standard criteria, and describe methods to analyze the impact of gene mutations on axonal transport in C. elegans. To obtain solid conclusions, it is necessary to image single neurons in vivo labeled by a specific promoter and to confirm that a mutation changes the localization of a cargo. The motility parameters of the transported cargo should then be analyzed in the mutant. This method enables the axonal transport of proteins and organelles, such as synaptic vesicle precursors and mitochondria, to be analyzed.
KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along ...microtubules. Mutations in human
lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established
models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our
models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.
Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this ...study, we compared the biochemical properties of two
kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.
KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations in KIF1A are associated with ...the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into the Caenorhabditis elegans KIF1A homolog unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.
KIF1A is a major axonal transport motor protein, but its functional significance remains elusive. Here we show that KIF1A-haploinsufficient mice developed sensory neuropathy. We found progressive ...loss of TrkA(+) sensory neurons in Kif1a+/− dorsal root ganglia (DRGs). Moreover, axonal transport of TrkA was significantly disrupted in Kif1a+/− neurons. Live imaging and immunoprecipitation assays revealed that KIF1A bound to TrkA-containing vesicles through the adaptor GTP-Rab3, suggesting that TrkA is a cargo of the KIF1A motor. Physiological measurements revealed a weaker capsaicin response in Kif1a+/− DRG neurons. Moreover, these neurons were hyposensitive to nerve growth factor, which could explain the reduced neuronal survival and the functional deficiency of the pain receptor TRPV1. Because phosphatidylinositol 3-kinase (PI3K) signaling significantly rescued these phenotypes and also increased Kif1a mRNA, we propose that KIF1A is essential for the survival and function of sensory neurons because of the TrkA transport and its synergistic support of the NGF/TrkA/PI3K signaling pathway.
•KIF1A haploinsufficiency caused sensory neuronal defects in mice•TrkA(+) nociceptive neurons were specifically decreased in Kif1a+/− DRGs•KIF1A specifically transports the NGF receptor TrkA to enhance PI3K signaling•In surviving neurons, PI3K-dependent sensitization of the pain receptor was impaired
How does the kinesin molecular motor contribute to the sensory nervous system? Using mouse molecular genetics, Tanaka et al. identified that KIF1A specifically transports the NGF receptor TrkA into axons of DRG neurons and that it serves in PI3K-mediated cell survival and sensitization.
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