Biology subjects

Shimomura, T.

Publications and source records attributed to Shimomura, T..

11 recordsLinked to original sources

bioRxiv2026

Enabling Stable Cortical Reconstruction in the HCP Pipeline at Standard Resolution Using FastSurfer Integration with Hybrid T2w- and T1w-based Masking

This study aimed to enable stable cortical reconstruction within the Human Connectome Project (HCP) pipeline under standard-resolution (1 mm3) conditions by developing an integrated framework combining FastSurfer with a hybrid T2w- and T1w-based masking strategy. We implemented a FastSurfer-integrated HCP pipeline incorporating a hybrid masking approach (t2log-hybrid) to stabilize cortical reconstruction, where SynthStrip-derived masks were refined using log-transformed T2-weighted and squared T1-weighted images. An anterior commissure-anchored spatial switching mechanism selectively replaced artifact-prone T2w regions with T1w-derived information in orbitofrontal areas. Performance was evaluated against FreeSurfer v6 (FS6), v7 (FS7), and FastSurfer using geometric agreement, regional consistency, and vertex-wise thickness-myelin analyses. The proposed framework reduced total processing time by over 50% compared with FS6 and maintained high geometric agreement with modern pipelines (vertex-wise r = 0.970 vs FastSurfer; r = 0.929 vs FS7). Agreement with FS6 was lower (r = 0.842), reflecting systematic differences in boundary definition rather than random error. The hybrid masking approach showed higher regional consistency and lower variability in T1w/T2w-derived myelin estimates (CV = 16.50% vs 40.56% in FastSurfer), with more spatially consistent thickness-myelin interaction patterns in artifact-prone ventral regions. Integrating FastSurfer into the HCP pipeline with hybrid T2w- and T1w-based masking enables stable cortical reconstruction by constraining signal-driven instability while preserving algorithm-dependent spatial organization. These findings highlight the importance of masking as a constraint on instability in cortical reconstruction and support the applicability of HCP-style analysis to clinical and legacy MRI datasets.

neuroscience↗

bioRxiv2025

Extracellular K+ modulates the pore conformations of Cys-loop receptor anion channels

K+ is an essential cation for life, but no eukaryotic membrane protein with a modulatory site for extracellular K+ has been discovered. Here, we report that a Cys-loop receptor, CG12344/DmAlka, expressed in the Drosophila nervous system, is selectively modulated by physiological concentration of extracellular K+. Structural prediction, electrophysiology and phylogenetic analysis of DmAlka revealed the extracellular K+ binding site that mimics the hydrated chemical environment for K+, as observed in K+ channel pore. Furthermore, we found that K+ binding induces a previously unrecognized "mode-switching," altering properties ranging from ligand sensitivity to ion selectivity. Notably, a human glycine receptor variant also exhibited similar mechanisms. Our study reveals a novel regulatory mechanism of Cys-loop receptors that directly links the extracellular K+ signaling to Cl- conductance in animals.

biophysics↗

bioRxiv2024

Characterization and engineering of a blue-sensitive, Gi/o-biased, and bistable ciliary opsin from a fan worm

Ciliary opsins have been identified not only in vertebrates but also in invertebrates. An invertebrate ciliary opsin was recently identified in the fan worm Acromegalomma interruptum (formerly named Megalomma interrupta); however, its spectral and signaling characteristics are unknown. In the present study, we characterized the spectral properties and light-induced cellular signaling properties of the opsin (AcrInvC-opsin). AcrInvC-opsin showed an absorption maximum at 464 nm and upon blue-light absorption, the spectrum was red-shifted by approximately 50 nm. The two states are inter-convertible by illumination with blue and orange light. Blue light illumination of AcrInvC-opsin caused specific coupling with Gi, sustained Gi dissociation, decreased intracellular cAMP levels, and activation of GIRK channels. The cellular responses by the activated opsin were partially terminated by orange light illumination. These light-dependent responses indicate that the InvC-opsin is a typical bistable pigment wherein the resting and activated states can be inter-converted by visible light illumination. We also attempted to modulate the spectral and functional properties of AcrInvC-opsin using site-directed mutagenesis. Substitution of Ser-94 with Ala caused little spectral shift in the resting state but a further red-shift of [~]10 nm in the activated state, indicating that the absorption spectra of the two states were tuned differently. In contrast, the S94A substitution did not significantly affect the light-dependent signaling properties of AcrInvC-opsin. Because AcrInvC-opsin is a blue-sensitive, Gi/o-biased, and bistable pigment, it has the potential to serve as an optical control tool to specifically and reversibly regulate Gi/o-dependent signaling pathways by visible light.

biophysics↗

bioRxiv2022

Conformational rearrangements in 2nd voltage sensor domain switch PIP2- and voltage-gating modes in two-pore channels

Two-pore channels (TPCs) are activated by PIP2 binding to domain I and/or by voltage-sensing in domain II (DII). Little is known about how these two stimuli are integrated and how each TPC subtype achieves its unique preference. Here, we show that the distinct conformations of DII-S4 in the voltage-sensor domain determine the two gating modes. DII-S4 takes an intermediate conformation, and forced stabilization in this conformation was found to give or maintain a high PIP2-dependence in primarily voltage- dependent TPC3 or in PIP2-gated and non-voltage-dependent TPC2, respectively. We also found in TPC2 that a tricyclic antidepressant desipramine induces the DII-S4 based voltage-dependence and that a flavonoid naringenin biases the mode preference from PIP2-gating to desipramine-induced voltage-gating. Taken together, our study on TPCs revealed an unprecedented mode-switching mechanism involving conformational changes in DII-S4. This will pave the way for drug development by targeting specific gating modes of TPCs. Significance statementMembrane voltage and PIP2 are different types of signals on endosomal and lysosomal membranes. The two signals are integrated into two-pore channels (TPCs) whose two repeating domains, DI and DII, play roles in PIP2 binding and voltage sensing, respectively. We showed that the conformation of the S4 helix in DII determines the voltage-dependent or PIP2-dependent gating mode, which explains the different preferences of the two signals between TPC subtypes. The preference for these two gating modes can be changed by a flavonoid, naringenin. Our findings on the molecular mechanism of the two gating modes in TPCs provide a clue to the understanding and pharmacological manipulation of the signaling by PIP2 and voltage in intracellular organelles.

biophysics↗

bioRxiv2021

A Simple Fluorescence Assay for Cystine Uptake via the xCT in Cells using Selenocystine and a Fluorescent Probe

The cystine/glutamate antiporter (xCT) is a crucial transporter that maintains cellular redox balance by regulating intracellular glutathione synthesis via cystine uptake. However, no robust and simple method to determine the cystine uptake activity of xCT is currently available. We have developed a method to measure the xCT activity via the reaction of selenocysteine and fluorescein O,O'-diacrylate (FOdA). Selenocystine, a cystine analog, is transported into cells through xCT on the cell membrane. The amount of the transported selenocystine was then determined by a reaction using tris(2-carboxyethyl)phosphine (TCEP) and FOdA in a weak acidic buffer at pH 6. Using this method, the cystine uptake activity of xCT in various cells, and the inhibitory efficiency of xCT inhibitors, were evaluated.

cell biology↗

bioRxiv2021

Intrinsic Gating Behavior of Voltage-Gated Sodium Channels Predetermines Regulation by Auxiliary β-subunits

Voltage-gated sodium (Nav) channels mediate rapid millisecond electrical signaling in excitable cells. Auxiliary subunits, {beta}1-{beta}4, are thought to regulate Nav channel function through covalent and/or polar interactions with the channel s voltage-sensing domains. How these interactions translate into the diverse and variable regulatory effects of {beta}-subunits remains unclear. Here, we find that the intrinsic movement order of the voltage-sensing domains during channel gating is unexpectedly variable across Nav channel isoforms. This movement order dictates the channel s propensity for closed-state inactivation, which in turn modulates the actions of {beta}1 and {beta}3. We show that the differential regulation of skeletal muscle, cardiac, and neuronal Nav channels is explained by their variable levels of closed-state inactivation. Together, this study provides a unified mechanism for the regulation of all Nav channel isoforms by {beta}1 and {beta}3, which explains how the fixed structural interactions of auxiliary subunits can paradoxically exert variable effects on channel function.

biophysics↗

bioRxiv2020

Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

Human ether-a-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. Although it is known that APETx1 inhibits hERG by stabilizing the resting state, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Here, we prepared a recombinant APETx1, which is structurally and functionally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, in addition to a previously reported acidic hERG residue, play key roles in the inhibition of hERG by APETx1. Docking models of the APETx1-VSD complex that satisfy the results of mutational analysis suggest a molecular recognition mode between APETx1 and the resting state of hERG; this would provide a structural basis for designing ligands that control hERG function by binding to the VSD.

molecular biology↗

bioRxiv2020

New Lipophilic Fluorescent Dyes for Exosome Labeling: Monitoring of Cellular Uptake of Exosomes

We have developed three types of exosomal membrane binding fluorescent probes, Mem Dye-Green, Mem Dye-Red and Mem Dye-Deep Red, to monitor exosome uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold, which allows for highly sensitive fluorescence imaging of the exosome. These dyes can also be used to observe the dynamics of exosomes in live cells. The use of PKH dyes (Figure 1), which are currently the most widely-used fluorescent probes for exosome labeling, has some limitations. For example, PKH dyes tend to aggregate to form exosome-like nanoparticles, and these nanoparticles are uptaken by cells. Moreover, Mehdi suggested that the use of PKH dyes triggers an enlargement of the exosome size owing to membrane fusion or intercalation. To overcome the limitations of PKH dyes, we introduce amphiphilic moieties to the cyanine. To investigate the characteristics of the Mem Dyes as exosome labeling probes, we perform nanoparticle tracking analysis (NTA), zeta potential measurement and confocal microscopy. The Mem Dyes show excellent performance for exosome labeling (no aggregation and less size shift). O_FIG O_LINKSMALLFIG WIDTH=91 HEIGHT=200 SRC="FIGDIR/small/931295v1_fig1.gif" ALT="Figure 1"> View larger version (8K): org.highwire.dtl.DTLVardef@e978aeorg.highwire.dtl.DTLVardef@1f208cdorg.highwire.dtl.DTLVardef@11fdb1dorg.highwire.dtl.DTLVardef@6a7373_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Chemical structure of PKH26. C_FIG

biochemistry↗

bioRxiv2019

A native prokaryotic voltage-dependent calcium channel with a novel selectivity filter sequence

Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling in living organisms. The structure of Cavs is similar to that of voltage-dependent Na+ channels (Navs). It is known that prokaryotic Navs can obtain Ca2+ selectivity by negative charge mutations of the selectivity filter, but native prokaryotic Cavs had not yet been identified.\n\nHere, we report the first identification of a native prokaryotic Cav, CavMr, and its relative, NavPp. Although CavMr contains a smaller number of negatively charged residues in the selectivity filter than artificial prokaryotic Cavs, CavMr exhibits high Ca2+ selectivity. In contrast, NavPp, which has similar filter sequence to artificial Cavs, mainly allows Na+ to permeate. Interestingly, a NavPp mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca2+ selectivity. Mutational analyses revealed that the glycine residue of the CavMr selectivity filter is a determinant for Ca2+ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs.\n\nThese findings provide new insight into the Ca2+ selectivity mechanism conserved from prokaryotes to eukaryotes.

biophysics↗

bioRxiv2018

In vitro characterization of multidrug-resistant influenza A(H1N1)pdm09 viruses carrying a dual amino acid substitution associated with reduced susceptibility to neuraminidase inhibitors

We detected influenza A(H1N1)pdm09 viruses carrying dual H275Y/I223R, H275Y/I223K, or H275Y/G147R substitutions in their neuraminidase protein, respectively. These viruses showed cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir. The H275Y/G147R virus retained its replication capability at least in vitro, but the H275Y/I223R and H275Y/I223K viruses did not.

microbiology↗

bioRxiv2018

SLO potassium channels antagonize premature decision making in C. elegans

SummaryAnimals have to modify their behavior at the right timing to respond to changes in environments. Yet, the molecular and neural mechanisms regulating the timing of behavioral transition remain largely unknown. Performing forward genetics on a plasticity of thermotaxis behavior in C. elegans, we demonstrated that SLO potassium channels together with a cyclic nucleotide-gated channel CNG-3 determine the timing of the transition of temperature preference after shift of cultivation temperature. We further revealed that SLO and CNG-3 channels regulate the alteration in responsiveness of thermosensory neurons. Our results suggest that the regulation of sensory adaptation is a major determinant of the latency for animals to make decisions in changing behavior.\n\nHighlightsO_LISlo-1 and SLO-2 K+ channels decelerated transition of temperature preference in thermotaxis behavior after upshift of cultivation temperature\nC_LIO_LISLO K+ channels slowed down the adaptation of AFD thermosensory neuron to new cultivation temperature\nC_LIO_LIA cyclic nucleotide-gated channel CNG-3 functioned together with SLO-2\nC_LIO_LIThermotaxis serves as could be a model system for early onset epilepsies\nC_LI

neuroscience↗