Publications by authors named "Shinsuke Niwa"

29 Publications

  • Page 1 of 1

Effects of dynein inhibitor on the number of motor proteins transporting synaptic cargos.

Biophys J 2021 Feb 19. Epub 2021 Feb 19.

Frontier Research Institute for Interdisciplinary Sciences and Graduate School of Life Science, Tohoku University, Sendai, Japan.

Synaptic cargo transport by kinesin and dynein in hippocampal neurons was investigated by non-invasively measuring the transport force based on non-equilibrium statistical mechanics. Although direct physical measurements such as force measurement using optical tweezers are difficult in an intracellular environment, the non-invasive estimations enabled enumerating force producing units (FPUs) carrying a cargo comprising the motor proteins generating force. The number of FPUs served as a barometer for stable and long-distance transport by multiple motors, which was then used to quantify the extent of damage to axonal transport by dynarrestin, a dynein inhibitor. We found that dynarrestin decreased the FPU for retrograde transport more than for anterograde transport. This result indicates the applicability of the non-invasive force measurements. In the future, these measurements may be used to quantify damage to axonal transport resulting from neuronal diseases including Alzheimer's, Parkinson's, and Huntington's diseases.
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http://dx.doi.org/10.1016/j.bpj.2021.02.018DOI Listing
February 2021

Neural and behavioral control in by a yellow-light-activatable caged compound.

Proc Natl Acad Sci U S A 2021 Feb;118(6)

Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, 113-0033 Tokyo, Japan;

is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist -vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.
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http://dx.doi.org/10.1073/pnas.2009634118DOI Listing
February 2021

A Combinatorial MAP Code Dictates Polarized Microtubule Transport.

Dev Cell 2020 04 27;53(1):60-72.e4. Epub 2020 Feb 27.

Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA. Electronic address:

Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.
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http://dx.doi.org/10.1016/j.devcel.2020.01.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181406PMC
April 2020

Going Too Far Is the Same as Falling Short: Kinesin-3 Family Members in Hereditary Spastic Paraplegia.

Front Cell Neurosci 2019 26;13:419. Epub 2019 Sep 26.

Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada.

Proper intracellular trafficking is essential for neuronal development and function, and when any aspect of this process is dysregulated, the resulting "transportopathy" causes neurological disorders. Hereditary spastic paraplegias (HSPs) are a family of such diseases attributed to over 80 spastic gait genes (SPG), specifically characterized by lower extremity spasticity and weakness. Multiple genes in the trafficking pathway such as those relating to microtubule structure and function and organelle biogenesis are representative disease loci. Microtubule motor proteins, or kinesins, are also causal in HSP, specifically mutations in Kinesin-I/KIF5A (SPG10) and two kinesin-3 family members; KIF1A (SPG30) and KIF1C (SPG58). KIF1A is a motor enriched in neurons, and involved in the anterograde transport of a variety of vesicles that contribute to pre- and post-synaptic assembly, autophagic processes, and neuron survival. KIF1C is ubiquitously expressed and, in addition to anterograde cargo transport, also functions in retrograde transport between the Golgi and the endoplasmic reticulum. Only a handful of KIF1C cargos have been identified; however, many have crucial roles such as neuronal differentiation, outgrowth, plasticity and survival. HSP-related kinesin-3 mutants are characterized mainly as loss-of-function resulting in deficits in motility, regulation, and cargo binding. Gain-of-function mutants are also seen, and are characterized by increased microtubule-on rates and hypermotility. Both sets of mutations ultimately result in misdelivery of critical cargos within the neuron. This likely leads to deleterious cell biological cascades that likely underlie or contribute to HSP clinical pathology and ultimately, symptomology. Due to the paucity of histopathological or cell biological data assessing perturbations in cargo localization, it has been difficult to positively link these mutations to the outcomes seen in HSPs. Ultimately, the goal of this review is to encourage future academic and clinical efforts to focus on "transportopathies" through a cargo-centric lens.
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http://dx.doi.org/10.3389/fncel.2019.00419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775250PMC
September 2019

Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors.

Proc Natl Acad Sci U S A 2019 09 27;116(37):18429-18434. Epub 2019 Aug 27.

Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, 980-0845 Miyagi, Japan;

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 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 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 mutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into the homolog 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.
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http://dx.doi.org/10.1073/pnas.1905690116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744892PMC
September 2019

Vital roles of PCNA K165 modification during C. elegans gametogenesis and embryogenesis.

DNA Repair (Amst) 2019 10 11;82:102688. Epub 2019 Aug 11.

Graduate School of Life Sciences, Tohoku University, Sendai, Japan; Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan. Electronic address:

In eukaryotes, the DNA damage bypass pathway is promoted by ubiquitylation of PCNA at the conserved lysine 164. Using CRISPR-Cas9 system, we introduced amino acid substitution at K165 of C. elegans PCNA that corresponds to K164 in other characterised organisms and examined the contribution of this residue at a variety of stages during development. In the presence of UV-induced DNA lesions, PCNA-K165 is crucial for not only the early embryonic stages but also during larval development, implicating its functions for a broad time period during animal development. We also show that, without induction of DNA damage, concomitant inhibition of PCNA ubiquitylation and checkpoint activation causes abnormal gametogenesis events and severely impairs reproduction of worms. Our findings suggest a conserved function of PCNA ubiquitylation in tolerance of UV-induced damage and also propose that PCNA ubiquitylation contributes to gametogenesis during unperturbed C. elegans development.
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http://dx.doi.org/10.1016/j.dnarep.2019.102688DOI Listing
October 2019

Fluorescence-labeled neopeltolide derivatives for subcellular localization imaging.

Org Biomol Chem 2019 07;17(28):6771-6776

Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan.

Design, synthesis and functional analysis of fluorescent derivatives of neopeltolide, an antiproliferative marine macrolide, are reported herein. Live cell imaging using the fluorescent derivatives showed rapid cellular uptake and localization within the endoplasmic reticulum as well as the mitochondria.
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http://dx.doi.org/10.1039/c9ob01276aDOI Listing
July 2019

Physical parameters describing neuronal cargo transport by kinesin UNC-104.

Biophys Rev 2019 Jun 21;11(3):471-482. Epub 2019 May 21.

Frontier Research Institute for Interdisciplinary Sciences (FRIS) and Graduate School of Life Sciences, Tohoku University, Sendai, Japan.

In this review, we focus on the kinesin-3 family molecular motor protein UNC-104 and its regulatory protein ARL-8. UNC-104, originally identified in Caenorhabditis elegans (C. elegans), has a primary role transporting synaptic vesicle precursors (SVPs). Although in vitro single-molecule experiments have been performed to primarily investigate the kinesin motor domain, these have not addressed the in vivo reality of the existence of regulatory proteins, such as ARL-8, that control kinesin attachment to/detachment from cargo vesicles, which is essential to the overall transport efficiency of cargo vesicles. To quantitatively understand the role of the regulatory protein, we review the in vivo physical parameters of UNC-104-mediated SVP transport, including force, velocity, run length and run time, derived from wild-type and arl-8-deletion mutant C. elegans. Our future aim is to facilitate the construction of a consensus physical model to connect SVP transport with pathologies related to deficient synapse construction caused by the deficient UNC-104 regulation. We hope that the physical parameters of SVP transport summarized in this review become a useful guide for the development of such model.
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http://dx.doi.org/10.1007/s12551-019-00548-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558098PMC
June 2019

KIF1Bβ mutations detected in hereditary neuropathy impair IGF1R transport and axon growth.

J Cell Biol 2018 10 20;217(10):3480-3496. Epub 2018 Aug 20.

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

KIF1Bβ is a kinesin-3 family anterograde motor protein essential for neuronal development, viability, and function. KIF1Bβ mutations have previously been reported in a limited number of pedigrees of Charcot-Marie-Tooth disease type 2A (CMT2A) neuropathy. However, the gene responsible for CMT2A is still controversial, and the mechanism of pathogenesis remains elusive. In this study, we show that the receptor tyrosine kinase IGF1R is a new direct binding partner of KIF1Bβ, and its binding and transport is specifically impaired by the Y1087C mutation of KIF1Bβ, which we detected in hereditary neuropathic patients. The axonal outgrowth and IGF-I signaling of neurons were significantly impaired, consistent with decreased surface IGF1R expression. The complementary capacity of KIF1Bβ-Y1087C of these phenotypes was significantly impaired, but the binding capacity to synaptic vesicle precursors was not affected. These data have supported the relevance of KIF1Bβ in IGF1R transport, which may give new clue to the neuropathic pathogenesis.
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http://dx.doi.org/10.1083/jcb.201801085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168269PMC
October 2018

A homozygous loss-of-function mutation causes growth delay, frequent seizures and severe intellectual disability.

Elife 2018 05 22;7. Epub 2018 May 22.

Institute of Medical Biology, Immunos, Singapore.

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in . The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme., CAMK2A failed to rescue neuronal defects in lacking , the ortholog of human , neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.
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http://dx.doi.org/10.7554/eLife.32451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963920PMC
May 2018

Streptothricin acetyl transferase 2 (Sat2): A dominant selection marker for Caenorhabditis elegans genome editing.

PLoS One 2018 9;13(5):e0197128. Epub 2018 May 9.

Division of Developmental Dynamics, Graduate School of Life Science Tohoku University, Aoba-ku, Sendai, Japan.

Studies on Caenorhabditis elegans would benefit from the introduction of new selectable markers to allow more complex types of experiments to be conducted with this model animal. We established a new antibiotic selection marker for C. elegans transformation based on nourseothricin (NTC) and its resistance-encoding gene, streptothricin-acetyl transferase 2 (Sat2). NTC was able to efficiently prevent worm development at very low concentrations, and the worms expressing Sat2 were able to survive on the selection plates without any developmental defects. Using CRISPR/Cas9 and NTC selection, we were able to easily insert a 13-kb expression cassette into a defined locus in C. elegans. The structure and spectrum of NTC differs from other antibiotics like hygromycin B and geneticin, making it possible to use NTC alongside them. Indeed, we confirmed NTC-sat2 selection could work with the hygromycin B selection system simultaneously. Thus, the new NTC-Sat2 system can act as a useful dominant marker for gene transfer and genome editing in C. elegans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197128PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942822PMC
August 2018

Non-invasive force measurement reveals the number of active kinesins on a synaptic vesicle precursor in axonal transport regulated by ARL-8.

Phys Chem Chem Phys 2018 Jan;20(5):3403-3410

Department of Applied Physics, Graduate School of Engineering, Tohoku University, Sendai, Japan.

Kinesin superfamily protein UNC-104, a member of the kinesin-3 family, transports synaptic vesicle precursors (SVPs). In this study, the number of active UNC-104 molecules hauling a single SVP in axons in the worm Caenorhabditis elegans was counted by applying a newly developed non-invasive force measurement technique. The distribution of the force acting on a SVP transported by UNC-104 was spread out over several clusters, implying the presence of several force-producing units (FPUs). We then compared the number of FPUs in the wild-type worms with that in arl-8 gene-deletion mutant worms. ARL-8 is a SVP-bound arf-like small guanosine triphosphatase, and is known to promote unlocking of the autoinhibition of the motor, which is critical for avoiding unnecessary consumption of adenosine triphosphate when the motor does not bind to a SVP. There were fewer FPUs in the arl-8 mutant worms. This finding indicates that a lack of ARL-8 decreased the number of active UNC-104 motors, which then led to a decrease in the number of motors responsible for SVP transport.
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http://dx.doi.org/10.1039/c7cp05890jDOI Listing
January 2018

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons.

Authors:
Shinsuke Niwa

J Vis Exp 2017 10 18(128). Epub 2017 Oct 18.

Frontier Research Institute for Interdisciplinary Sciences and Graduate School of Life Sciences, Tohoku University;

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.
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http://dx.doi.org/10.3791/56690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752426PMC
October 2017

Structural basis for CRMP2-induced axonal microtubule formation.

Sci Rep 2017 09 6;7(1):10681. Epub 2017 Sep 6.

RIKEN Center for Life Science Technologies, Tsurumi-ku, Yokohama, 230-0045, Japan.

Microtubule associated protein Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity in developing neurons through interactions with tubulins or microtubules. However, how CRMP2 promotes axonal formation by affecting microtubule behavior remains unknown. This study aimed to obtain the structural basis for CRMP2-tubulin/microtubule interaction in the course of axonogenesis. The X-ray structural studies indicated that the main interface to the soluble tubulin-dimer is the last helix H19 of CRMP2 that is distinct from the known C-terminal tail-mediated interaction with assembled microtubules. In vitro structural and functional studies also suggested that the H19-mediated interaction promoted the rapid formation of GTP-state microtubules directly, which is an important feature of the axon. Consistently, the H19 mutants disturbed axon elongation in chick neurons, and failed to authorize the structural features for axonal microtubules in Caenorhabditis elegans. Thus, CRMP2 induces effective axonal microtubule formation through H19-mediated interactions with a soluble tubulin-dimer allowing axonogenesis to proceed.
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http://dx.doi.org/10.1038/s41598-017-11031-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587665PMC
September 2017

BORC Regulates the Axonal Transport of Synaptic Vesicle Precursors by Activating ARL-8.

Curr Biol 2017 Sep 17;27(17):2569-2578.e4. Epub 2017 Aug 17.

Howard Hughes Medical Institute, Department of Biology, Stanford University, 385 Serra Mall, Stanford, CA 94305, USA; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China. Electronic address:

Axonal transport of synaptic vesicle precursors (SVPs) is essential for synapse development and function. The conserved ARF-like small GTPase ARL-8 is localized to SVPs and directly activates UNC-104/KIF1A, the axonal-transport kinesin for SVPs in C. elegans. It is not clear how ARL-8 is activated in this process. Here we show that part of the BLOC-1-related complex (BORC), previously shown to regulate lysosomal transport, is required to recruit and activate ARL-8 on SVPs. We found mutations in six BORC subunits-blos-1/BLOS1, blos-2/BLOS2, snpn-1/Snapin, sam-4/Myrlysin, blos-7/Lyspersin, and blos-9/MEF2BNB-cause defects in axonal transport of SVPs, leading to ectopic accumulation of synaptic vesicles in the proximal axon. This phenotype is suppressed by constitutively active arl-8 or unc-104 mutants. Furthermore, SAM-4/Myrlysin, a subunit of BORC, promotes the GDP-to-GTP exchange of ARL-8 in vitro and recruits ARL-8 onto SVPs in vivo. Thus, BORC regulates the axonal transport of synaptic materials and synapse formation by controlling the nucleotide state of ARL-8. Interestingly, the other two subunits of BORC essential for lysosomal transport, kxd-1/KXD1 and blos-8/Diaskedin, are not required for the SVP transport, suggesting distinct subunit requirements for lysosomal and SVP trafficking.
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http://dx.doi.org/10.1016/j.cub.2017.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693321PMC
September 2017

Autoinhibition of a Neuronal Kinesin UNC-104/KIF1A Regulates the Size and Density of Synapses.

Cell Rep 2016 08 11;16(8):2129-2141. Epub 2016 Aug 11.

Howard Hughes Medical Institute, Department of Biology, Stanford University, 385 Serra Mall, Stanford, CA 94305, USA. Electronic address:

Kinesin motor proteins transport intracellular cargoes throughout cells by hydrolyzing ATP and moving along microtubule tracks. Intramolecular autoinhibitory interactions have been shown for several kinesins in vitro; however, the physiological significance of autoinhibition remains poorly understood. Here, we identified four mutations in the stalk region and motor domain of the synaptic vesicle (SV) kinesin UNC-104/KIF1A that specifically disrupt autoinhibition. These mutations augment both microtubule and cargo vesicle binding in vitro. In vivo, these mutations cause excessive activation of UNC-104, leading to decreased synaptic density, smaller synapses, and ectopic localization of SVs in the dendrite. We also show that the SV-bound small GTPase ARL-8 activates UNC-104 by unlocking the autoinhibition. These results demonstrate that the autoinhibitory mechanism is used to regulate the distribution of transport cargoes and is important for synaptogenesis in vivo.
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http://dx.doi.org/10.1016/j.celrep.2016.07.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432123PMC
August 2016

The nephronophthisis-related gene ift-139 is required for ciliogenesis in Caenorhabditis elegans.

Authors:
Shinsuke Niwa

Sci Rep 2016 08 12;6:31544. Epub 2016 Aug 12.

Frontier Research Institute for Interdisciplinary Sciences and Graduate School of Life Sciences, Tohoku University, Aramaki Aza Aoba 6-3, Aobaku, Sendai, Miyagi 980-8578, Japan.

Defects in cilia cause a spectrum of diseases known as ciliopathies. Nephronophthisis, a ciliopathy, is the most common genetic cause of renal disease. Here, I cloned and analysed a nephronophthisis-related gene ift-139 in Caenorhabditis elegans. ift-139 was exclusively expressed in ciliated neurons in C. elegans. Genetic and cellular analyses suggest that ift-139 plays a role in retrograde intraflagellar transport and is required for cilia formation. A homologous point mutation that causes ciliopathy disrupted the function of ift-139 in C. elegans. ift-139 is an orthologue of human TTC21B, mutations in which are known to cause nephronophthisis 12 and short-rib thoracic dysplasia 4. These results suggest that ift-139 is evolutionarily conserved and fundamental to the formation of cilia.
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http://dx.doi.org/10.1038/srep31544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981862PMC
August 2016

The Molecular Motor KIF1A Transports the TrkA Neurotrophin Receptor and Is Essential for Sensory Neuron Survival and Function.

Neuron 2016 06 2;90(6):1215-1229. Epub 2016 Jun 2.

Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Center of Excellence in Genome Medicine Research, King Abdulaziz University, Jeddah 21589, Saudi Arabia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neuron.2016.05.002DOI Listing
June 2016

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

J Neurosci 2015 Mar;35(12):5067-86

Department of Molecular and Cell Biology and Department of Molecular Structure and Dynamics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan, and Center of Excellence in Genome Medicine Research, King Abdulaziz University, Jeddah 21589, Saudi Arabia

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.
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http://dx.doi.org/10.1523/JNEUROSCI.4240-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705379PMC
March 2015

Kinesin superfamily proteins and the regulation of microtubule dynamics in morphogenesis.

Authors:
Shinsuke Niwa

Anat Sci Int 2015 Jan 28;90(1):1-6. Epub 2014 Oct 28.

Department of Biological Sciences, Stanford University, 385 Serra Mall, Herrin Lab 144, Stanford, CA, 94305, USA,

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.
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http://dx.doi.org/10.1007/s12565-014-0259-5DOI Listing
January 2015

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

EMBO J 2013 May 15;32(10):1352-64. Epub 2013 Mar 15.

Department of Cell Biology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β-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 β-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 β-tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.
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http://dx.doi.org/10.1038/emboj.2013.59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655465PMC
May 2013

A molecular motor, KIF13A, controls anxiety by transporting the serotonin type 1A receptor.

Cell Rep 2013 Feb 8;3(2):509-19. Epub 2013 Feb 8.

Department of Cell Biology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.

Molecular motors are fundamental to neuronal morphogenesis and function. However, the extent to which molecular motors are involved in higher brain functions remains largely unknown. In this study, we show that mice deficient in the kinesin family motor protein KIF13A (Kif13a(-/-) mice) exhibit elevated anxiety-related behavioral phenotypes, probably because of a reduction in 5HT(1A) receptor (5HT(1A)R) transport. The cell-surface expression level of the 5HT(1A)R was reduced in KIF13A-knockdown neuroblastoma cells and Kif13a(-/-) hippocampal neurons. Biochemical analysis showed that the forkhead-associated (FHA) domain of KIF13A and an intracellular loop of the 5HT(1A)R are the interface between the motor and cargo vesicles. A minimotor consisting of the motor and FHA domains is able to transport 5HT(1A)R-carrying organelles in in vitro reconstitution assays. Collectively, our results suggest a role for this molecular motor in anxiety control.
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http://dx.doi.org/10.1016/j.celrep.2013.01.014DOI Listing
February 2013

KIF19A is a microtubule-depolymerizing kinesin for ciliary length control.

Dev Cell 2012 Dec 15;23(6):1167-75. Epub 2012 Nov 15.

Department of Cell Biology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Cilia control homeostasis of the mammalian body by generating fluid flow. It has long been assumed that ciliary length-control mechanisms are essential for proper flow generation, because fluid flow generation is a function of ciliary length. However, the molecular mechanisms of ciliary length control in mammals remain elusive. Here, we suggest that KIF19A, a member of the kinesin superfamily, regulates ciliary length by depolymerizing microtubules at the tips of cilia. Kif19a(-/-) mice displayed hydrocephalus and female infertility phenotypes due to abnormally elongated cilia that cannot generate proper fluid flow. KIF19A localized to cilia tips, and recombinant KIF19A controlled the length of microtubules polymerized from axonemes in vitro. KIF19A had ATP-dependent microtubule-depolymerizing activity mainly at the plus end of microtubules. Our results indicated a molecular mechanism of ciliary length regulation in mammals, which plays an important role in the maintenance of the mammalian body.
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http://dx.doi.org/10.1016/j.devcel.2012.10.016DOI Listing
December 2012

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

Proc Natl Acad Sci U S A 2012 Jan 17;109(5):1725-30. Epub 2012 Jan 17.

Department of Cell Biology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

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.
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http://dx.doi.org/10.1073/pnas.1107808109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277188PMC
January 2012

Preferential binding of a kinesin-1 motor to GTP-tubulin-rich microtubules underlies polarized vesicle transport.

J Cell Biol 2011 Jul 18;194(2):245-55. Epub 2011 Jul 18.

Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.

Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively accumulates at the tips of axons. However, it remains unknown what cue KIF5 recognizes to result in this selective accumulation. We found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.
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http://dx.doi.org/10.1083/jcb.201104034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144414PMC
July 2011

Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease.

Neuron 2010 Nov;68(4):610-38

Departments of Cell Biology and Anatomy and Molecular Structure and Dynamics, Graduate School of Medicine, University of Tokyo, Hongo, Tokyo 113-0033, Japan.

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.
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http://dx.doi.org/10.1016/j.neuron.2010.09.039DOI Listing
November 2010

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

Cell 2009 Nov;139(4):802-13

Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

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.
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http://dx.doi.org/10.1016/j.cell.2009.10.023DOI Listing
November 2009

Kinesin superfamily motor proteins and intracellular transport.

Nat Rev Mol Cell Biol 2009 Oct;10(10):682-96

Department of Cell Biology and Anatomy, University of Tokyo, Japan.

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.
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http://dx.doi.org/10.1038/nrm2774DOI Listing
October 2009

KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD.

Nat Cell Biol 2008 Nov 12;10(11):1269-79. Epub 2008 Oct 12.

Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.

Synaptic proteins are synthesized in the cell body and transported down the axon by microtubule-dependent motors. We previously reported that KIF1Bbeta and KIF1A motors are essential for transporting synaptic vesicle precursors; however the mechanisms that regulate transport, as well as cargo recognition and control of cargo loading and unloading remain largely unknown. Here, we show that DENN/MADD (Rab3-GEP) is an essential part of the regulation mechanism through direct interaction with the stalk domain of KIF1Bbeta and KIF1A. We also show that DENN/MADD binds preferentially to GTP-Rab3 and acts as a Rab3 effector. These molecular interactions are fundamental as sequential genetic perturbations revealed that KIF1Bbeta and KIF1A are essential for the transport of DENN/MADD and Rab3, whereas DENN/MADD is essential for the transport of Rab3. GTP-Rab3 was more effectively transported than GDP-Rab3, suggesting that the nucleotide state of Rab3 regulates axonal transport of Rab3-carrying vesicles through preferential interaction with DENN/MADD.
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http://dx.doi.org/10.1038/ncb1785DOI Listing
November 2008