Biology subjects

Sakurai, T.

Publications and source records attributed to Sakurai, T..

28 records · Page 2Linked to original sources

bioRxiv2023

The CaliAli tool for long-term tracking of neuronal population dynamics in calcium imaging

Neuron-tracking algorithms exhibit suboptimal performance in calcium imaging when the same neurons are not consistently detected, as unmatched features hinder intersession alignment. CaliAli addresses this issue by employing an alignment-before-extraction strategy that incorporates vasculature information to improve the detectability of weak signals and maximize the number of trackable neurons. By excelling in neural remapping and high spatial overlap scenarios, CaliAli paves the way toward further understanding long-term neural network dynamics.

neuroscience↗

bioRxiv2023

Linear contraction of stress fibers generates cell body rotation

Wounds are healed by crawling migration of the epidermal cells around the injured area. Fish epidermal keratocytes that rapidly repair wounds comprise a frontal crescent-shaped lamellipodium and a rear rugby ball-shaped cell body. The cell body rotates like a wheel during migration. Stress fibers, which are bundles of contractile actomyosin filaments, are arranged along the seams of the rugby ball. Here we show the linear contraction of stress fibers to be the driving force for rotation. We constructed a mechanical model of the cell body that consisted of a soft cylinder with a contractile coil. From the motion of the model, it was predicted that contraction of the stress fibers would deform the soft cell body, as a result of which the deformed cell body would push against the substrate to generate torque. This prediction was confirmed by the observation of stress fiber dynamics in migrating cells. Linear-to-rotation conversion in migrating keratocytes is realized by simple soft-body mechanics. Conversion from linear motion to rotation is widely used in machines with moving parts, but requires somewhat complicated mechanics. An understanding of linear-to-rotation conversion in keratocytes has potential for use in the design of biomimetic soft robots.

biophysics↗

bioRxiv2022

High-throughput platform for depolarization-induced Ca2+ release for skeletal muscle disease mutation validation and drug discovery

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including {beta}1a, Stac3 adaptor protein and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for validation of mutations and drug discovery for skeletal muscle diseases. SummaryMutations in essential components for depolarization-induced Ca2+ release (DICR) are implicated into various skeletal muscle diseases. Murayama et al. establish a reconstituted DICR platform in nonmuscle cells for evaluation of disease-causing mutations and drug discovery.

physiology↗

bioRxiv2021

Enhanced CICR activity reduces ER Ca2+ level in cells expressing CPVT-linked mutant RyR2

Type 2 ryanodine receptor (RyR2) is a cardiac Ca2+ release channel in the endoplasmic reticulum (ER). Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), which is considered to be associated with enhanced spontaneous Ca2+ release. This spontaneous Ca2+ release tends to occur when ER Ca2+ ([Ca2+]ER) reaches a certain threshold level, and CPVT mutations are reported to lower this threshold. There are two explanations for this lowered threshold: the mutations increase sensitivity to luminal Ca2+ or they enhance cytosolic [Ca2+] ([Ca2+]cyt)-induced Ca2+ release (CICR) activity. However, no quantitative analysis of this issue has been performed so far. Here, we quantitatively explored how the change in CICR activity of RyR2 affects the threshold [Ca2+]ER experimentally and by model-based simulation. Wild-type (WT) and CPVT-linked mutant RyR2s were expressed in HEK293 cells. [Ca2+]cyt and [Ca2+]ER measurements with Ca2+ indicators revealed that CPVT RyR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER in a mutation-specific manner compared with WT cells. The CICR activity of mutant RyR2s was assessed by Ca2+-dependent [3H]ryanodine binding and parameter analysis. CICR activity at resting [Ca2+]cyt, A7.0, was higher in CPVT mutants than in WT and a strong inverse correlation was found between threshold [Ca2+]ER and A7.0. Interestingly, lowering RyR2 expression increased threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on net Ca2+ release rate via RyR2, a product of A7.0 for each mutant and the density of RyR2 molecules. A model-based simulation successfully reproduced the [Ca2+]cyt and [Ca2+]ER changes. Interestingly, the CICR activity associated with specific mutations correlated well with the age of onset of the disease in CPVT patients carrying the mutations. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced CICR activity without considering a change in the [Ca2+]ER sensitivity of RyR2. SummaryCPVT-linked RyR2 mutations are prone to induce spontaneous Ca2+ release from ER, which is strongly associated with arrhythmias. Kurebayashi et al. quantitatively explore how the changes in CICR activity by RyR2 mutations affect spontaneous Ca2+ experimentally and by model simulation.

physiology↗

bioRxiv2021

Pheromone binding protein shapes olfactory temporal resolution

Male moths are capable of orienting towards conspecific females using sex pheromones. Since pheromones are distributed as discontinuous plumes owing to air turbulence, tracking intermittent stimuli with high temporal resolution is suggested to be important for efficient localisation. Here, using a pheromone binding protein (BmPBP1) knockout silkmoth, we revealed that the loss of functional pheromone binding protein altered antennal response kinetics resulting in reduced temporal resolution to intermittent pheromone stimuli on the antennae. Behavioural analysis revealed that BmPBP1-knockout males exhibited significantly less straight walking, which occurs when detecting pheromone stimuli, especially to high frequency stimuli. Accordingly, BmPBP1-knockout males took a significantly longer time to locate pheromone sources and females than did wild-type males. Together, BmPBP1 plays a critical role in determining temporal antennal response kinetics and that an appropriate range of temporal sensory and behavioural resolutions is essential for tracking pheromone plumes for efficient pheromone source localisation in the silkmoth.

neuroscience↗

bioRxiv2020

Gating mechanism of cardiac ryanodine receptor 2 upon calcium ion binding

Cardiac ryanodine receptor (RyR2) is a large Ca2+ release channel in the sarcoplasmic reticulum and indispensable for excitation-contraction coupling in the heart. RyR2 is activated by Ca2+ and RyR2 mutations are implicated in severe arrhythmogenic diseases. Yet, the structural basis underlying channel opening and how mutations affect the channel remain unknown. Here, we addressed gating mechanism of RyR2 by combining high-resolution structures determined by cryo-electron microscopy with quantitative functional analysis of channels carrying various mutations in specific residues. We demonstrated two fundamental mechanisms for channel gating: interactions close to the channel pore stabilize the channel to prevent hyperactivity and a series of interactions in the surrounding regions is necessary for channel opening upon Ca2+ binding. Mutations at the residues involved in the former and the latter mechanisms cause gain-of-function and loss-of-function, respectively. Our results reveal gating mechanisms of the RyR2 channel and alterations by pathogenic mutations at the atomic level.

biochemistry↗

bioRxiv2020

A novel saline-soluble, rapidly-metabolized RyR1 inhibitor rescues volatile anesthesia-induced death and environmental heat stroke in a mouse model relevant to malignant hyperthermia

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduced resting intracellular Ca2+, inhibited halothane- and isoflurane-induced Ca2+ release, suppressed caffeine-induced contracture in skeletal muscle, reduced sarcolemmal cation influx, and prevented or reversed the fulminant MH crisis induced by isoflurane anesthesia and rescued animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

pharmacology and toxicology↗

bioRxiv2019

The Setaria viridis genome and diversity panel enables discovery of a novel domestication gene

Diverse wild and weedy crop relatives hold genetic variants underlying key evolutionary innovations of crops under domestication. Here, we provide genome resources and probe the genetic basis of domestication traits in green millet (Setaria viridis), a close wild relative of foxtail millet (S. italica). Specifically, we develop and exploit a platinum-quality genome assembly and de novo assemblies for 598 wild accessions to identify loci underlying a) response to climate, b) a key loss of shattering trait that permits mechanical harvest, and c) leaf angle, a major predictor of yield in many grass crops. With CRISPR-Cas9 genome editing, we validated Less Shattering1 (SvLES1) as a novel gene for seed shattering, which is rendered non-functional via a retrotransposon insertion in SiLes1, the domesticated loss-of-shattering allele of S. italica. Together these results and resources project S. viridis as a key model species for complex trait dissection and biotechnological improvement of panicoid crops.

genomics↗

bioRxiv2018

Domain organization and conformational change of dynactin p150

Dynactin is a principal regulator of the minus-end directed microtubule motor dynein. The sidearm of dynactin is essential for binding to microtubules and regulation of dynein activity. Although our understanding of the structure of the dynactin backbone (Arp1 rod) has greatly improved recently, structural details of the sidearm part remain elusive. Here, electron microscopy of individual molecules of the dynactin complex revealed that the sidearm was highly flexible and exhibited diverse morphologies. Utilizing mutants for nanogold labeling and deletion analysis, we determined the domain organization of the largest subunit p150 and identified a filamentous structure protruding from the head domain of the sidearm as the coiled-coil 1 (CC1), the dynein-binding domain, in p150. Furthermore, the protrusion formed by CC1 exhibited either a folded or an extended form, suggesting that CC1 works as an extending \"arm\". These findings provide clues to understand how dynactin binds to microtubules and regulates dynein.

biophysics↗

bioRxiv2018

Activation of lateral hypothalamic group III mGluRs suppresses drug-seeking following abstinence and cocaine-associated increases in excitatory drive to orexin/hypocretin cells

The perifornical/lateral hypothalamic area (LHA) orexin (hypocretin) system is involved in drug-seeking behavior elicited by drug-associated stimuli. Cocaine exposure is associated with presynaptic plasticity at LHA orexin cells such that excitatory input to orexin cells is enhanced, both acutely and into withdrawal. These changes may augment orexin cell reactivity to drug-related cues during abstinence and contribute to relapse-like behavior. Studies in hypothalamic slices from drug-naive animals indicate that agonism of group III metabotropic glutamate receptors (mGluRs) reduces presynaptic glutamate release onto orexin cells. Therefore, we examined the group III mGluR system as a potential target to reduce orexin cell excitability in-vivo, and tested whether activating these receptors could normalize orexin cell activity following cocaine and reduce cocaine-seeking elicited by drug-associated stimuli during abstinence. First, we verified that group III mGluRs regulate orexin cell activity in vivo by showing that intra-LHA infusions of the selective agonist L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) reduces Fos expression in orexin cells following 24h food deprivation. Next, we extended these findings to show that intra-LHA L-AP4 infusions reduced discriminative stimulus-driven cocaine-seeking following withdrawal. L-AP4 had no effect on general motor activity of sucrose self-administration. Finally, using whole-cell patch clamp recordings from identified orexin cells in orexin-GFP transgenic mice, we show that enhanced presynaptic drive to orexin cells persists for up to 14d into withdrawal and that this plasticity is normalized by L-AP4. L-AP4 had no effect on measures of postsynaptic plasticity in cocaine-exposed animals. Together, these data indicate that agonism of LHA group III mGluRs reduces orexin cell activity in-vivo and is an effective strategy to suppress cocaine-seeking behavior following withdrawal. These effects are likely mediated, at least in part, by normalization of presynaptic plasticity at orexin cells that occurs as a result of cocaine exposure.

neuroscience↗