Biology subjects

Kita, T.

Publications and source records attributed to Kita, T..

12 recordsLinked to original sources

bioRxiv2025

Horizontally Tiled Network of Cortico-Basal Ganglia Modules Performs Reinforcement Learning

The neocortex and basal ganglia nuclei are connected along regions that share the same topography and are arranged side-by-side. Inspired by the anatomical characteristics of the cerebrum, we developed a network in which the modules of the neocortex-basal ganglia unit were arranged in a horizontally tiled manner. By applying this network to reinforcement learning tasks, we demonstrated that reinforcement learning can be achieved through horizontal signals passing between modules. Each module not only performs its calculation but also provides signals to adjacent modules. This lateral transmission takes advantage of the differences in the projection ranges of the three basal ganglia pathways, the direct, indirect, and hyperdirect pathways, which have been examined in physiologic studies. We found that these differences enabled temporal-difference-error computations. This study proposes a novel strategy for information processing based on neocortical-basal ganglia circuits, highlighting the computational significance of their anatomically and physiologically clarified features.

neuroscience↗

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

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↗

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

Transcriptomic Landscape of Hyperthyroidism in Mice Overexpressing Thyroid Stimulating Hormone

Hyperthyroidism is a condition with excessive thyroid hormone secretion. Activation of thyroid stimulating hormone receptor (TSHR) fundamentally leads to hyperthyroidism. The details of TSHR signaling remain to be elucidated. We conducted transcriptome analyses for hyperthyroid mice that we generated by overexpressing TSH. TSH overexpression via hydrodynamic gene delivery with pLIVE-TSHB and pLIVE-CGA vectors consistently caused hyperthyroidism and goiters for at least 4 weeks in C57BL/6J mice. RNA sequencing analysis of their thyroid glands revealed that thiamazole slightly changed the thyroid transcriptome, which reinforces a conventional theory that thiamazole decreases thyroid hormone secretion via inhibition of thyroid peroxidase activity. Meanwhile, TSH overexpression drastically changed the thyroid transcriptome. In particular, enrichment analyses identified the cell cycle, phosphatidylinositol-3 kinase/Akt pathway, and Ras-related protein 1 pathway as possibly associated with goiter development. Regarding the role of TSHR signaling in hyperthyroidism, it is noteworthy that Slc26a4 was exclusively upregulated among genes crucial to thyroid hormone secretion at both 1 and 4 weeks after hydrodynamic gene delivery. To verify the relationship between this upregulation and hyperthyroidism, we overexpressed TSH in Slc26a4 knockout mice. TSH overexpression caused hyperthyroidism in Slc26a4 knockout mice, equivalent to that in control mice. To summarize, we analyzed hyperthyroid mice generated by TSH overexpression. We did not observe significant changes in known genes and pathways involved in thyroid hormone secretion. Thus, our datasets might include candidate genes that have not yet been identified as regulators of thyroid function. Our transcriptome datasets regarding hyperthyroidism can contribute to future research on TSHR signaling.

physiology↗

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

Single-cell RNA-seq reveals stepwise fate conversion of supporting cells to hair cells in the chick auditory epithelium

In contrast to mammals, the avian cochlea, specifically the basilar papilla, can regenerate sensory hair cells, which involves fate conversion of supporting cells to hair cells. To determine the mechanisms for converting supporting cells to hair cells, we used single-cell RNA sequencing during hair cell regeneration in explant cultures of chick basilar papillae. We identified dynamic changes in the gene expression of supporting cells, and the pseudotime trajectory analysis demonstrated the stepwise fate conversion from supporting cells to hair cells. Initially, supporting cell identity was erased and transition to the precursor state occurred. A subsequent gain in hair cell identity progressed together with downregulation of precursor-state genes. Transforming growth factor beta receptor 1-mediated signaling was involved in induction of the initial step, and its inhibition resulted in suppression of hair cell regeneration. Our data provide new insights for understanding fate conversion from supporting cells to hair cells in avian basilar papillae.

neuroscience↗

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

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↗