Biology subjects

Niwa, S.

Publications and source records attributed to Niwa, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Kif2C safeguards radial glial integrity to prevent cortical malformation

Radial glial cells (RGCs) generate cortical neurons and guide radial neuronal migration, yet how microtubule (MT) regulators coordinate progenitor maintenance, mitotic fidelity, and cortical architecture remains unclear. Here, we identify the MT depolymerase Kif2C/MCAK as an essential regulator of RGC integrity with relevance to human neurodevelopmental disorders. Kif2C is enriched in developing cortical RGCs and localizes to radial fibers, basal endfeet, and mitotic structures. Acute Kif2C depletion in embryonic mouse cortices disrupts RG fiber organization, impairs neuronal migration, reduces the RGC pool, and induces mitotic defects, chromosome segregation errors, DNA damage, and cell-cycle arrest. Kif2C-deficient cortices further exhibit focal pial basement membrane disruption and neuronal overmigration, resulting in a cobblestone-like cortical malformation. We identify two individuals with neurodevelopmental disorders carrying rare deleterious KIF2C variants and show that a patient-derived truncating variant fails to rescue Kif2C-deficient cortical phenotypes, implicating KIF2C dysfunction in human neurodevelopmental disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/726143v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1970844org.highwire.dtl.DTLVardef@9fe29eorg.highwire.dtl.DTLVardef@d72ddcorg.highwire.dtl.DTLVardef@103f729_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

The KASH protein UNC-83 differentially regulates kinesin-1 activity to control developmental stage-specific nuclear migration

Nuclear migration plays a fundamental role in development, requiring precise spatiotemporal control of bidirectional movement through dynein and kinesin motors. Here, we uncover a mechanism for developmental regulation of nuclear migration directionality. The nuclear envelope KASH protein UNC-83 in Caenorhabditis elegans exists in multiple isoforms that differentially control motor activity. The shorter UNC-83c isoform promotes kinesin-1-dependent nuclear movement in embryonic hyp7 precursors, while longer UNC-83a/b isoforms facilitate dynein-mediated nuclear migration in larval P cells. We demonstrate that UNC-83as N-terminal domain functions as a kinesin-1 inhibitory module by directly binding kinesin heavy chain (UNC-116). This isoform-specific inhibition, combined with differential affinity for kinesin light chain (KLC-2), establishes a molecular switch for directional control. Together, these interdisciplinary studies reveal how alternative isoforms of cargo adaptors can generate developmental stage-specific regulation of motor activity during development.

cell biology↗

bioRxiv2025

KLP-6 is a kinesin superfamily protein resistant to ADP inhibition

Product inhibition is a type of enzyme inhibition in which the reaction product suppresses further enzyme activity. Kinesins are microtubule-dependent ATPases that move along microtubules by hydrolyzing ATP into ADP and inorganic phosphate (Pi). Like other ATPases, kinesins are inhibited by their hydrolysis product, ADP. We show here that Caenorhabditis elegans kinesin-3, KLP-6, is a unique kinesin that is resistant to ADP inhibition. Amino acid sequence comparisons between KLP-6 and KIF1A, another kinesin-3, revealed that KLP-6 possesses a unique sequence in an 2a helix. Substituting this region with the corresponding sequence from KIF1A abolished KLP-6s markedly higher binding selectivity for ATP over ADP, confirming that this domain is crucial for its insensitivity to ADP inhibition. Molecular dynamics simulations uncovered that the 2a helix of KLP-6 adopts a less stable helical conformation in the ADP-bound state than in the ATP-bound state, which explains KLP-6s strong selectivity for ATP. Our results provide valuable insights into kinesins nucleotide selectivity and underlying molecular mechanism.

biochemistry↗

bioRxiv2024

Modular photostable fluorescent DNA blocks dissect the effects of pathogenic mutant kinesin on collective transport

Intracellular transport is driven by teams of various motor proteins. Advances in DNA nanotechnology have enabled the programmable linkage of different types of motor proteins. In this study, we developed a modular, photostable, fluorescence-labeled tiny DNA origami block (FTOB) for extended observation of collective transport by selected motor combinations. The FTOB is designed as a 4-helix bundle ([~]8.4 nm) with densely accumulated fluorescent dyes, minimizing blinking and photobleaching. By designing a pair of connector DNAs, FTOBs are heterodimerized following motor protein attachment using the ALFA-tag/nanobody system. Our system examined the impact of a pathogenic mutant kinesin on its collective movement with wild-type kinesin, clearly observing two distinct behaviors: the teams velocity was generally governed by the slower mutant but occasionally surged to levels comparable to that of a single wild-type motor. Our photostable, robust, modular FTOB system could serve as a versatile tool for precisely dissecting cooperative cargo transport.

bioengineering↗

bioRxiv2024

Dimerization GAS2 mediates microtubule and F-actin crosslinking

GAS2 was originally identified as a growth arrest-specific protein, and recent studies have revealed its involvement in multiple cellular processes. Its dual interaction with actin filaments and microtubules highlights its essential role in cytoskeletal organization, such as cell division, apoptosis, and possibly tumorigenesis. However, the structural bases by which GAS2 regulates cytoskeletal dynamics remain unclear. In this study, we present cryo-EM structures of the GAS2- CH3 domain in complex with F-actin at 2.8 [A] resolution, representing the first type 3 CH domain structure bound to F-actin, confirming its actin-binding activity. We also provide the first near- atomic resolution cryo-EM structure of the GAS2-GAR domain bound to microtubules and identified conserved microtubule-binding residues. Our biochemical experiments show that GAS2 promotes microtubule nucleation and polymerization and its C-terminal region is essential for dimerization, bundling of both F-actin and microtubules, and microtubule nucleation. Based on these results, we propose how GAS2 controls cytoskeletal organization.

biochemistry↗

bioRxiv2024

Processivity of the monomeric KLP-6 kinesin and a Brownian ratchet model with symmetric potentials

Most kinesin molecular motors dimerize to move processively and efficiently along microtubules; however, some can maintain processivity even in a monomeric state. Previous studies have suggested that asymmetric potentials between the motor domain and microtubules underlie this motility. In this study, we demonstrate that the kinesin-3 family motor protein KLP-6 can move along microtubules as a monomer upon release of autoinhibition. This motility can be explained by a change in length between the head and tail, rather than by asymmetric potentials. Using mass photometry and single-molecule assays, we confirmed that activated full-length KLP-6 is monomeric both in solution and on microtubules. KLP-6 possesses a microtubule-binding tail domain, and its motor domain does not exhibit biased movement, indicating that the tail domain is crucial for the processive movement of monomeric KLP-6. We developed a mathematical model to explain the unidirectional movement of monomeric KLP-6. Our model concludes that a slight conformational change driven by neck-linker docking in the motor domain enables the monomeric kinesin to move unidirectionally if a second microtubule-binding domain exists. SIGNIFICANCEKinesin molecular motors are designed to move efficiently using two heads. Studying these biological molecular motors provides valuable insights into the mechanisms that generate unidirectional movements amidst intense thermal fluctuations. This study reveals that the monomeric kinesin-3 KLP-6 can move along microtubules through interactions with its tail domain. The proposed Brownian ratchet model explains this movement by considering a change in stalk length caused by neck-linker docking rather than asymmetric potentials. This model suggests that a slight conformational change can achieve robust processive movement of kinesin. These findings have significant implications for understanding Brownian ratchet motors and designing rational artificial molecular motors.

biophysics↗

bioRxiv2024

The chromokinesin Kid forms a homodimer and moves processively along microtubules

During prometaphase in mitosis, chromosomes are pushed toward the spindle equator. The chromokinesin Kid, also known as KIF22, moves chromosomes along spindle microtubules during prometaphase. Kid has long been considered a monomeric and non-processive motor, different from typical kinesins. In this study, we demonstrate that the full-length Kid forms a homodimer and moves processively along microtubules. A conserved coiled-coil domain within the stalk region of Kid is sufficient for homodimer formation and is required for the processivity of Kid. Furthermore, the neck linker and coiled-coil domains of Kid could add processive activity to the motor domain of KIF1A, suggesting that Kid contains a functional neck linker and dimerization capability, a prerequisite for the processivity of kinesin motor domains. The full-length Kid, containing a helix-hairpin-helix domain, can transport double-stranded DNA along microtubules in vitro. Alphafold3 prediction suggests that the dimerization of Kid stabilizes the association with DNA. These findings collectively suggest the reclassification of Kid as a processive and dimeric motor that transports DNA along microtubules.

biochemistry↗

bioRxiv2024

SLC-25A46 Regulates Mitochondrial Fusion through FZO-1/Mitofusin and is Essential for Maintaining Neuronal Morphology

Mitochondria are dynamic organelles shaped by sequential fission and fusion events. The mitochondrial protein SLC25A46 has been identified as a causative gene for mitochondrial neuropathies. However, the function of SLC25A46 in mitochondrial morphogenesis remains controversial, with several reports suggesting it acts as a mitochondrial fission factor, while others propose it as a fusion factor. In this study, employing forward genetics, we identified slc-25A46, a Caenorhabditis elegans orthologue of human SLC25A46, as an essential factor for mitochondrial fusion. Suppressor mutagenesis screening revealed loss-of-function mutations in drp-1, a mitochondrial fission factor, as suppressors of slc-25A46. The phenotype of slc-25A46 mutants is similar to those of fzo-1 mutants, wherein the mitochondrial fusion factor Mitofusin is disrupted. Overexpressing FZO-1/Mitofusin mitigated mitochondrial defects in slc-25a46 mutants, indicating SLC-25A46 promotes fusion through FZO-1/Mitofusin. Disease model worms carrying mutations associated with SLC25A46 exhibited mitochondrial fragmentation and accelerated neurodegeneration, suggesting slc-25A46 maintains neuronal morphology through mitochondrial fusion regulation.

cell biology↗

bioRxiv2023

The FHA domain is essential for the autoinhibition of KIF1A/UNC-104

KIF1A/UNC-104, a member of the kinesin super family motor proteins, plays a pivotal role in the axonal transport of synaptic vesicles and their precursors. Drosophila melanogaster UNC-104 (DmUNC-104) is a relatively recently discovered Drosophila kinesin. Although some point mutations that disrupt synapse formation have been identified, the biochemical properties of DmUNC-104 protein have not been investigated. Here, we prepared recombinant full-length DmUNC-104 protein and determined the biochemical features. We analyzed the effect of a previously identified missense mutation in the FHA domain, called bristly (bris), and found that wild-type DmUNC-104 is a mixture of monomer and dimer and the bris mutation strongly promote the dimerization. Furthermore, we found that disease-associated mutations also promote the dimerization of DmUNC-104. These findings suggest that the FHA domain is essential for the autoinhibition of KIF1A/UNC-104, and that abnormal dimerization of KIF1A is linked to human diseases.

biochemistry↗

bioRxiv2023

Characterizing Human KIF1Bβ Motor Activity by Single-Molecule Motility Assays and Caenorhabtidis elegans Genetics.

The axonal transport of synaptic vesicle precursors relies on KIF1A and UNC-104 ortholog motors. In mammals, KIF1B{beta} is also responsible for the axonal transport of synaptic vesicle precursors. Mutations in KIF1A and KIF1B{beta} lead to a wide range of neuropathies. While previous studies have revealed the biochemical, biophysical and cell biological properties of KIF1A, and its defects in neurological disorders, the fundamental properties of KIF1B{beta} remain elusive. In this study, we determined the motile parameters of KIF1B{beta} through single-molecule motility assays. Additionally, we established simple methods for testing the axonal transport activity of human KIF1B{beta} using Caenorhabditis elegans genetics. Taking advantage of these methods, we demonstrated that these assays enable the detection of reduced KIF1B{beta} activities both in vitro and in vivo, that is caused by a disease-associated mutation.

cell biology↗

bioRxiv2023

Kinesin family member Kif23 regulates cytokinetic division and maintains neural stem/progenitor cell pool in the developing neocortex

Accurate mitotic division of neural stem cell/progenitor cells (NSPCs) is crucial for the coordinated generation of progenitors and neurons in the developing cortex. Here, we investigated the pivotal role of Kif23, an N-kinesin motor protein, in embryonic mouse NSPCs. We found that Kif23 is highly expressed in the mitotic NSPCs within the embryonic cortex of both mouse and human. Knockdown (KD) of Kif23 led to precocious neurogenesis, attributed to an accelerated cell cycle exit, likely resulting from disrupted mitotic spindle orientation and impaired cytokinesis. Kif23 KD induced upregulation of the {gamma}-H2AX-p53-p21 signaling pathway, ultimately culminating in cytokinetic failure. Additionally, Kif23 depletion perturbed the apical surface structure of NSPCs and disrupted the proper localization of apical junctional proteins. Importantly, we demonstrated the successful rescue of Kif23 KD-induced phenotypes by introducing wild-type human KIF23, but not by a variant of KIF23 with a microcephaly-associated mutation. Our findings underscore the critical role of Kif23 in cortical development and provide novel insights into the intricate molecular mechanisms underlying pathogenesis of microcephaly. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/564302v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@5488e0org.highwire.dtl.DTLVardef@1e65146org.highwire.dtl.DTLVardef@1e1ec76org.highwire.dtl.DTLVardef@16b7477_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Modeling the motion of disease-associated KIF1A heterodimers

KIF1A is a member of the kinesin-3 family motor protein that transports synaptic vesicle precursors in axons. Mutations in the Kif1a gene cause neuronal diseases. Most patients are heterozygous and have both mutated and intact KIF1A alleles, suggesting that heterodimers composed of wild-type KIF1A and mutant KIF1A are likely involved in pathogenesis. In this study, we propose mathematical models to describe the motility of KIF1A heterodimers composed of wild-type KIF1A and mutant KIF1A. Our models precisely describe run length, run time, and velocity of KIF1A heterodimers using a few parameters obtained from two homodimers. The independent head model is a simple hand-over-hand model in which stepping and detachment rates from a microtubule of each head are identical to those in the respective homodimers. Although the velocities of heterodimers expected from the independent head model were in good agreement with the experimental results, this model underestimated the run lengths and run times of some heterodimeric motors. To address this discrepancy, we propose the coordinated head model, in which we hypothesize a tethered head, in addition to a microtubule-binding head, contributes to microtubule binding in a vulnerable one-head-bound state. The run lengths and run times of the KIF1A heterodimers predicted by the coordinated head model matched well with experimental results, suggesting a possibility that the tethered head affects the microtubule binding of KIF1A. Our models provide insights into how each head contributes to the processive movement of KIF1A and can be used to estimate motile parameters of KIF1A heterodimers. SIGNIFICANCEKIF1A is responsible for transporting synaptic vesicle precursors in axons. KIF1A mutations are associated with neurodegener-ative diseases. Most of these mutations are de novo and autosomal dominant, suggesting that half of the motors in patients are heterodimers composed of wild-type and mutant KIF1A. However, reliable theoretical models to explain the behavior of heterodimeric motors are lacking. In this study, we obtained exact analytical solutions to describe run length, run time, and velocity of heterodimeric motors which move in a hand-over-hand fashion. Our models provide valuable tools for quantitatively understanding the impact of heterodimerization with mutant KIF1A and the cooperative behavior of KIF1A dimers.

biophysics↗

bioRxiv2023

Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3

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 Caenorhabditis elegans 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 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.

cell biology↗

bioRxiv2022

Diversification of the recombinant anti-kinesin monoclonal antibody H2

Kinesin-1, a motor protein composed of the kinesin heavy chain (KHC) and the kinesin light chain (KLC), is fundamental to cellular morphogenesis and function. A monoclonal antibody (mAb) called H2 recognizes the KHC in a broad range of species and is one of the most widely used mAbs in cytoskeletal motor research. Here, we generated vectors that expressed recombinant H2 in mammalian cells. We demonstrated that the recombinant H2 performed as well as the hybridoma-derived H2 in western blotting and immunofluorescence assays. The recombinant H2 could detect all three human KHC isotypes (KIF5A, KIF5B, and KIF5C) and amyotrophic lateral sclerosis (ALS)-associated KIF5A aggregates in the cell. Immunofluorescence microscopy showed that the single chain variable fragment (scFv) derived from the H2 mAb could specifically recognize KHCs in cells. In addition, we developed a chickenized anti-KHC scFv(H2), which broadens the application of H2 in immunofluorescence microscopy. Collectively, our findings validate recombinant H2 as useful for studying the function of KHCs.

cell biology↗

bioRxiv2022

ALS-associated mutant KIF5A forms oligomers and aggregates and induces neuronal toxicity

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({Delta}exon27), is predisposed to form oligomers and aggregates in cultured mouse cell lines. Interestingly, purified KIF5A({Delta}exon27) oligomers showed more active movement on microtubules than wild type KIF5A in vitro. Purified KIF5A({Delta}exon27) was prone to form aggregates in vitro. Moreover, KIF5A({Delta}exon27)-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.

cell biology↗

bioRxiv2022

Structural model of microtubule dynamics inhibition by Kinesin-4 from the crystal structure of KLP-12 - tubulin complex

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

biophysics↗

bioRxiv2021

Extreme value analysis of intracellular cargo transport by motor proteins

Extreme-value analysis (EVA) deals with deviations in the data from the median of the probability distributions. EVA serves various purposes such as predicting disasters and analyzing sports records. Herein, we extended the use of EVA to investigate the motility functions of nanoscale motor proteins in neurons of the living worm Caenorhabditis elegans (C. elegans). Motor proteins, such as kinesin and dynein, move along microtubules anterogradely and retrogradely, respectively, to deliver the cargo-containing materials needed for the cells. Although the essential difference between the two motors could not be inferred from the mean velocity values, the return-level EVA plots obtained from the velocity data revealed a difference. Shape parameters of the generalized extreme value distribution of EVA{xi} < 0 for anterograde transport and{xi} [≥] 0 for retrograde transport. Our findings extend the possibility and applicability of EVA for analyzing motility data of nanoscale proteins in vivo.

biophysics↗

bioRxiv2021

De novo disease-associated mutations in KIF1A dominant negatively inhibit axonal transport of synaptic vesicle precursors

KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A when they are dimerized. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND caused by loss of function mutations have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. Suppressor screening using the disease model worm identified a mutation that recovers the motor activity of disease-associated human KIF1A. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations. Significance StatementKIF1A is a molecular motor that transports synaptic vesicle precursors in axons. Recent studies have identified many KIF1A mutations in congenital neuropathy patients; however, the molecular mechanism of pathogenesis remains largely elusive. This study established loss of function models for KIF1A-associated neuronal disorder (KAND) in Caenorhabditis elegans to analyze the molecular and cell biology of the disease in vivo. Genetic screening using the disease model could find a mutation that recovers the motor activity of disease-associated KIF1A. This study also established in vitro single-molecule assays to quantitatively analyze the effect of KAND mutations when mutant KIF1A forms heterodimers with wild-type KIF1A. Our findings provide a foundation for future genetic screening and for drug screening to search for KAND treatments.

cell biology↗